Oligomeric compounds and compositions for use in modulation of pri-miRNAs

ABSTRACT

Compounds, compositions and methods are provided for modulating the levels expression, processing and function of pri-miRNAs. In particular, methods and compounds are provided for the modulation of the levels, expression, processing or function of polycistronic pri-miRNAs. The compositions comprise oligomeric compounds targeted to small non-coding RNAs and pri-miRNAs. Further provided are methods for selectively modulating pri-miRNA levels in a cell. Also provided are methods for identifying oligomeric compounds that result in increase pri-miRNA levels when contacted with a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/329,992 filedJan. 10, 2006, now U.S. Pat. No. 7,759,319 which is acontinuation-in-part of U.S. application Ser. No. 10/909,125, filed Jul.30, 2004, now U.S. Pat. No. 7,683,036 which claims priority under 35U.S.C. §119(e) to U.S. provisional applications Ser. No. 60/492,056filed Jul. 31, 2003, Ser. No. 60/516,303 filed Oct. 31, 2003, Ser. No.60/531,596 filed Dec. 19, 2003, and Ser. No. 60/562,417 filed Apr. 14,2004, each which is incorporated herein by reference in its entirety;and also claims priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication No.: 60/612,831 filed on Jan. 10, 2005, which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

A paper copy of the sequence listing and a computer-readable form of thesequence listing, on diskette, containing the file named CORE0016USP1SEQ.txt, which was created on Jan. 10, 2006, are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulationof small non-coding RNAs, particularly pri-miRNAs. In particular, thisinvention relates to compounds, particularly oligomeric compounds,which, in some embodiments, hybridize with or sterically interfere withnucleic acid molecules comprising or encoding small non-coding RNAtargets, including pri-miRNAs.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are small (approximately 21-22 nucleotides in length,these are also known as “mature” miRNA), non-coding RNA moleculesencoded in the genomes of plants and animals. These highly conserved,endogenously expressed RNAs regulate the expression of genes by bindingto the 3′-untranslated regions (3′-UTR) of specific mRNAs. MiRNAs mayact as key regulators of cellular processes such as cell proliferation,cell death (apoptosis), metabolism, and cell differentiation. On alarger scale, miRNA expression has been implicated in early development,brain development, disease progression (such as cancers and viralinfections). There is speculation that in higher eukaryotes, the role ofmiRNAs in regulating gene expression could be as important as that oftranscription factors. More than 200 different miRNAs have beenidentified in plants and animals (Ambros et al., Curr. Biol., 2003, 13,807-818). Mature miRNAs appear to originate from long endogenous primarymiRNA transcripts (also known as pri-miRNAs, pri-mirs, pri-miRs orpri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee,et al., EMBO J., 2002, 21(17), 4663-4670).

The current model of miRNA processing involves primary miRNA transcriptsbeing processed by a nuclear enzyme in the RNase III family known asDrosha, into approximately 70 nucleotide-long pre-miRNAs (also known asstem-loop structures, hairpins, pre-mirs or foldback miRNA precursors)which are subsequently processed by the Dicer RNase into mature miRNAs,approximately 21-25 nucleotides in length. It is believed that, inprocessing pri-miRNA into the pre-miRNA, the Drosha enzyme cutspri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang(Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, PlantPhysiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee,et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003,425, 415-419). The 3′ two-nucleotide overhang structure, a signature ofRNaseIII cleavage, has been identified as a critical specificitydeterminant in targeting and maintaining small RNAs in the RNAinterference pathway (Murchison, et al., Curr. Opin. Cell. Biol., 2004,16, 223-9). Both the primary RNA transcripts (pri-miRNAs) and foldbackmiRNA precursors (pre-miRNAs) are believed to be single-stranded RNAmolecules with at least partial double-stranded character, oftencontaining smaller, local internal hairpin structures. In someinstances, primary miRNA transcripts are processed such that onesingle-stranded mature miRNA molecule is generated from one arm of thehairpin-like structure of pri-miRNA; such primary miRNA transcripts areoften referred to as monocistronic pri-miRNA transcripts. Alternatively,a pri-miRNA transcript contains multiple hairpin structures, anddifferent hairpins give rise to different miRNAs. These are consideredpolycistronic miRNA transcripts, and each hairpin containing a maturemiRNA is given a unique gene name and the miRNA present on a singletranscript may be referred to as a “cluster” of such miRNAs. Examples ofpolycistronic miRNA clusters include the miR-17-92 cluster and themiR-15/miR-16-1 cluster.

Functional analyses of miRNAs have revealed that these small non-codingRNAs contribute to different physiological processes in animals,including developmental timing, organogenesis, differentiation,patterning, embryogenesis, growth control and programmed cell death.Examples of particular processes in which miRNAs participate includestem cell differentiation, neurogenesis, angiogenesis, hematopoiesis,and exocytosis (reviewed by Alvarez-Garcia and Miska, Development, 2005,132, 4653-4662).

Links between miRNAs, including miRNA families and clusters, and humandisease have been also been identified. Many miRNAs are de-regulated inprimary human tumors (Calin et al., Proc. Natl. Acad. Sci, 2002, 99,15524-15529; Calin et al., Proc. Natl. Acad. Sci, 2004, 101,11755-11760; He et al., Nature, 2005, 435, 828-833; Lu et al., Nature,2005, 435, 834). Moreover, many human miRNAs are located at genomicregions linked to cancer (Calin et al., Proc. Natl. Acad. Sci, 2004,101, 2999-3004; McManus, 2003, Semin. Cancer Biol, 13, 252-258; He etal., Nature, 2005, 435, 828-833). Mir-15a and mir-16-1, which arederived from a polycistronic miRNA, are located within a 30-kb regionchromosome 13q14, a region deleted in more than half of B cell chroniclymphocytic leukemias (B-CLL). Both mir-15a and mir-16-1 are deleted ordown-regulated in the majority of CLL cases (Calin et al., Proc. Nat.Acad. Sci, 2002, 99, 15524-15529).

Families of miRNAs are characterized by nucleotide identity at positions2-8 of the miRNA, a region known as the seed sequence. Lewis et al.describe several miRNA families, as well as miRNA superfamilies, whichare characterized by related seed sequences (Lewis et al. 2005).

MiRNAs are thought to exercise post-transcriptional control in mosteukaryotic organisms and have been detected in plants and animals aswell as certain viruses. A large number of miRNAs have been identifiedfrom several species (see for example PCT Publication WO 03/029459 andPublished US Patent Applications 20050222399, 20050227934, 20050059005and 20050221293) and many more have been bioinformatically predicted.Many of these miRNA are conserved across species, but species specificmiRNA have also been identified (Pillai, RNA, 2005, 11, 1753-1761).

Small non-coding RNA-mediated regulation of gene expression is anattractive approach to the treatment of diseases as well as infection bypathogens such as bacteria, viruses and prions and other disordersassociated with RNA expression or processing. By way of example,modulating the expression or processing of miR-122 may present anapproach for antiviral therapies, studies of a genetic interactionbetween miR-122 and the 5′ noncoding region of the hepatitis C viralgenome suggest that miR-122 is likely to facilitate replication of thehepatitis C viral RNA (Jopling, et al., Science, 2005, 5740, 1577-1581).

Consequently, there is a need for agents that regulate gene expressionvia the mechanisms mediated by small non-coding RNAs. Identification ofoligomeric compounds that can increase or decrease gene expression oractivity by modulating the levels of miRNA in a cell is thereforedesirable.

The present invention therefore provides oligomeric compounds andmethods useful for modulating the levels, expression, or processing ofpri-miRNAs, including those relying on mechanisms of action such as RNAinterference and dsRNA enzymes, as well as antisense and non-antisensemechanisms. One having skill in the art, once armed with this disclosurewill be able, without undue experimentation, to identify compounds,compositions and methods for these uses.

SUMMARY OF THE INVENTION

The present invention provides oligomeric compounds, especially nucleicacid and nucleic acid-like oligomeric compounds, which are targeted to,or mimic, nucleic acids comprising or encoding small non-coding RNAs,and which act to modulate the levels of small non-coding RNAs,particularly pri-miRNAs, or interfere with their function.

The present invention also provides oligomeric compounds, especiallynucleic acid and nucleic acid-like oligomeric compounds, which aretargeted to pri-miRNAs, and which act to modulate the levels ofpri-miRNAs, or interfere with their processing or function. The presentinvention further provides oligomeric compounds that target a regionflanking or overlapping a Drosha recognition region within a pri-miRNA.Additionally, the present invention provides oligomeric compounds thattarget a region flanking or overlapping a Drosha cleavage site. Thepresent invention also provides oligomeric compounds that increaselevels of a pri-miRNA. For example, the present invention providesoligomeric compounds 15 to 30 nucleobases in length targeted to a Drosharecognition region within a polycistronic pri-miRNA transcript. Thepolycistronic pri-miRNA transcript can be that from which miR-15a andmiR-16-1 are derived. The Drosha recognition region can be that ofmiR-16-1. Such oligomeric compounds may be antisense oligonucleotides,and may contain one or more chemical modifications. Additionally, sucholigomeric compounds are capable of increasing pri-miR-15a andpri-miR-16-1 levels.

Also provided are methods of modulating the levels of small non-codingRNAs, particularly pri-miRNAs, in cells, tissues or animals comprisingcontacting said cells, tissues or animals with one or more of thecompounds or compositions of the invention.

Further provided are methods of modulating the levels of miRs derivedfrom a polycistronic pri-miR transcript in a cell comprising selecting apolycistronic pri-miR transcript, selecting a Drosha recognition regionof a single miRNA derived from the selected polycistronic pri-miRtranscript, selecting an oligomeric compound 15 to 30 nucleotides inlength targeted to or sufficiently complementary to the selected Drosharecognition region, and contacting the cell with the oligomericcompound. Such methods include modulating the levels of a single maturemiRNA derived from the selected polycistronic pri-miRNA, oralternatively modulating the levels of two or more mature miRNAs derivedfrom the selected polycistronic pri-miRNA. Also provided are methods ofmodulating the levels of pri-miRNA-15a and pri-miR-16-1 comprisingcontacting a cell with an oligomeric compound targeted to orsufficiently complementary to Drosha-recognition regions onpri-miRNA-16-1 or pri-miRNA-15a.

The present invention provides methods for selectively modulating asingle member of a miR family in a cell comprising selecting a member ofa miR family derived from a pri-miR transcript, identifying one or moreoligomeric compounds targeted to or sufficiently complementary to theDrosha recognition region of a the selected pri-miR transcript, whereinthe identified oligomeric compounds lack sufficient complementarity tothe Drosha recognition regions of pri-miR transcripts from which othermembers of the miR family are derived, and contacting the cell with suchan identified oligomeric compound.

The present invention also provides oligomeric compounds comprising afirst strand and a second strand wherein at least one strand contains amodification and wherein a portion of one of the oligomeric compoundstrands is capable of hybridizing to a small non-coding RNA targetnucleic acid.

The present invention also provides oligomeric compounds comprising afirst region and a second region and optionally a third region whereinat least one region contains a modification and wherein a portion of theoligomeric compound is capable of hybridizing to a small non-coding RNAtarget nucleic acid.

The present invention provides methods for identifying oligomericcompounds capable of modulating pri-miRNA levels. A pri-miRNA isselected, and oligomeric compounds are designed such that they aretargeted to or sufficiently complementary to various target segmentswithin a pri-miRNA sequence, including oligomeric compounds targeted toand overlapping the mature miRNA sequence within the pri-miRNA. Anincrease in the level of a pri-miRNA in cells contacted with theoligomeric compounds as compared to cells not contacted with theoligomeric compounds indicates that the oligomeric compound modulatesthe pri-miRNA level.

The present invention further provides methods for identifying smallmolecules capable of modulating pri-miRNA levels. A pri-miRNA isselected, and small molecules are evaluated for their ability ofmodulate pri-miRNA levels. The small molecules may bind to the regionsof the pri-miR containing or overlapping the mature miRNA sequence, orthe Drosha recognition region. An increase in the level of a pri-miRNAin cells contacted with the small molecules as compared to cells notcontacted with the small molecules indicates that the small moleculemodulates the pri-miRNA levels.

DETAILED DESCRIPTION

The present invention provides methods for modulating the levels,expression, processing or function of small non-coding RNAs,particularly pri-miRNAs. Such methods are useful for the modulation of asingle mature miRNA derived from a polycistronic pri-miRNA transcript.Alternatively, such methods are useful for the modulation of multiplemature miRNAs derived from a polycistronic pri-miRNA transcript. Apolycistronic pri-miRNA transcript is selected for modulation in a cell,and a Drosha recognition region on the selected polycistronic pri-miRNAis identified. The Drosha recognition region is that of a single miRNAderived from the polycistronic pri-miRNA transcript. One or moreoligomeric compounds are identified that are targeted to or sufficientlycomplementary to the identified Drosha recognition region, and a cell iscontacted with the oligomeric compound. The contacting of the cell withan oligomeric compound targeted to or sufficiently complementary to theidentified Drosha recognition region results in a modulation of thelevel of a pri-miRNA, as evidenced by a change in the level of thepri-miRNA as compared to the level in cells not contacted with theoligomeric compound. An increase or decrease in the pri-miRNA levelindicates that the oligomeric compound modulates the level, expression,or processing of the pri-miRNA.

As used herein, the term “polycistronic pri-miRNA transcript” or“polycistronic miRNA transcript” refers to a pri-miRNA transcriptcontaining multiple hairpin structures, each of which gives rise to adifferent miRNA; each hairpin is given a unique gene name. In oneaspect, miRNAs derived from a polycistronic miRNA transcript may bespatially or temporally coordinately expressed, i.e. they have similarexpression profiles in a particular cell or tissue or in a particularstage of cell growth or organism development, respectively. In analternative aspect, miRNAs derived from a polycistronic pri-miRNA arenot expressed in a coordinated manner. Examples of polycistronic miRNAclusters include, but are not limited to, the miR-17-92 cluster and themiR-15/miR-16-1 cluster.

As used herein, the term “miRNA cluster” refers to two or more miRNAswhose transcription and/or processing is controlled in a coordinatedmanner. The coordinated expression and/or processing of an miRNA clustermay be controlled spatially or temporally, i.e. controlled within aparticular tissue or cell type or during a particular stage of cellgrowth or organism development, respectively. Examples of miRNA clustersare known in the art and include, but are not limited to, miR-23/27/24-2containing three miRNAs; miR-17/18/19a/20/19b-1 containing five miRNAs;and miR-17-92 containing seven miRNAs. Additionally, miR-15a andmiR-16-1 comprise a miRNA cluster.

As used herein, the term “miRNA family” refers to a plurality of miRNAsthat are related by nucleotide sequence. Thus, the members of an miRNAfamily are also known as “related miRNAs”. Each member of a miRNA familyshares an identical seed sequence. As used herein, the term “seedsequence” refers to nucleotides 2 to 6 or 2 to 7 from the 5′-end of amature miRNA sequence. Examples of miRNA families are known in the artand include, but are not limited to, the let-7 family (having 9 miRNAs),the miR-15 family (comprising miR-15a, miR-15b, miR-16-1, and miR-195),and the miR-181 family (comprising miR-181a, miR-181b, and miR-181c).

Polycistronic miRNA transcript, miRNA clusters and miRNA families havebeen found to be aberrantly expressed in disease states, i.e. miRNAsderived from the a polycistronic miRNA transcript, or miRNAs that aremembers of miRNA families or clusters are collectively present at higheror lower levels in a diseased cell or tissue as compared to healthy cellor tissue. In one embodiment, a polycistronic miRNA transcript, a miRNAcluster, or an miRNA family that is aberrantly expressed in a diseasestate is selected for modulation using the oligomeric compounds andmethods of the present invention.

As used herein, the term “monocistronic pri-miRNA transcript” or“monocistronic miRNA transcript” refers to a pri-miRNA from which asingle pre-miRNA, and consequently a single miRNA, is derived. Examplesof monocistronic pri-miRNA transcripts include, but are not limited to,those transcripts containing miR-122, miR-21, miR-1, and miR-30a.

As used herein, the term “miRNA precursor” is used to encompass, withoutlimitation, primary RNA transcripts, pri-miRNAs, including polycistronicpri-miRNAs and monocistronic pri-miRNAs, and pre-miRNAs.

As used herein, the term “Drosha recognition region” within a pri-miRNAtranscript encompasses the mature miRNA as well as up to 25 nucleotidesin the 5′ direction relative to the 5′ Drosha cleavage site of suchmature miRNA, and up to 50 nucleotides in the 3′ direction relative tothe 3′ Drosha cleavage site of such mature miRNA. In additionalembodiments, the Drosha recognition region encompasses the mature miRNAand up to 15 nucleotides in the 5′ direction relative to the 5′ Droshacleavage site of such mature miRNA, and up to 40 nucleotides in the 3′direction relative to the 3′ Drosha cleavage site of such mature miRNA.In some aspects, the Drosha recognition region is a region stronglyaffected by oligomeric compounds targeted to this region, i.e. thetargeting of oligomeric compounds to this region of a pri-miRNA resultsin a greater than 3.5-fold increase in the level of the pri-miRNA. Inother aspects, the level of the pri-miRNA is moderately affected byoligomeric compounds targeted to this region, i.e. the targeting ofoligomeric compounds to this Drosha recognition region results in a 1.5to 2.5-fold increase in the levels of the pri-miRNA. In further aspects,the targeting of a pri-miRNA with an oligomeric compound of theinvention affects the processing of one or more miRNAs on apolycistronic pri-miRNA.

As used herein, the term “Drosha cleavage site” is used to refer to asite approximately 22 nucleobases from the junction of the terminalhairpin loop and the stem of a pri-miRNA. One end of the miRNA isdetermined by selection of the cleavage site by the Drosha enzyme.

In one embodiment, a polycistronic pri-miRNA transcript containing apreferentially processed miRNA is selected for modulation. miRNAs withina cluster or derived from a polycistronic pri-miRNA transcript mayexhibit different levels or patterns of expression. For example, a firstmiRNA derived from a polycistronic miRNA transcript may be a“preferentially processed miRNA”, i.e. an miRNA that is found in a cellat higher levels, than a second miRNA derived from the samepolycistronic miRNA transcript. Targeting a preferentially processedmiRNA or the Drosha recognition region of a preferentially processedmiRNA may result in accumulation of the entire pri-miRNA transcript,which in turn results in the reduction of the levels of multiple miRNAsderived from the polycistronic pri-miRNA transcript. In one non-limitingexample, within the miR-15 family, miR-16-1 is found at higher levelsthan miR-15a. Oligomeric compounds targeted to the pri-miR-16-1 Drosharecognition region result in increased pri-miR-15a and pri-miR-16-1levels, whereas oligomeric compounds targeted to or sufficientlycomplementary to the pri-miR-15a Drosha recognition region result in theincrease of pri-miR-15a. Preferentially processed miRNA may differ amongcell types, tissues, or developmental stages.

The present invention provides methods for the selective modulation of asingle member of a miRNA family in a cell. The presence of identicalseed sequences amongst miRNA family members may preclude theidentification of oligomeric compounds which are sufficientlycomplementary to hybridize to only a single member of the miRNA family.Thus, in the methods provided herein, an oligomeric compound is selectedto that is sufficiently complementary to the Drosha recognition regionof the selected miRNA, resulting in the reduction of pri-miRNA levels orinhibition of pri-miRNA processing, which in turn results in thereduction of the levels of the selected miRNA. A member of a miRNAfamily, derived from a polycistronic or monocistronic pri-miRNAtranscript, is selected for modulation. And oligomeric compound isidentified which is targeted to or sufficiently complementary to theDrosha recognition region of the selected miRNA and which also lackssufficient complementarity to the Drosha recognition regions of one ormore of the remaining members of the miRNA family. Contacting a cellwith the identified oligomeric compound results in the modulation of thesingle selected member of the miRNA family, while the remaining miRNAfamily members are not modulated.

The present invention provides methods of identifying a Drosharecognition region in a pri-miRNA. Oligomeric compounds are designedsuch that they are targeted to or sufficiently complementary to varioustarget segments within a pri-miRNA sequence, including oligomericcompounds targeted or overlapping the mature miRNA sequence within thepri-miRNA. An increase in the level of a pri-miRNA in cells contactedwith the oligomeric compounds as compared to cells not contacted withthe oligomeric compounds indicates that the oligomeric compoundmodulates pri-miRNA levels. As exemplified herein, the pri-miR-16-1Drosha recognition region encompasses: the mature miR-16-1 sequence; upto 25 nucleotides in the 5′ direction relative to the 5′ Drosha cleavagesite within pri-miR-16-1; and up to 50 nucleotides in the 3′ directionrelative to the 3′ Drosha cleavage site within pri-miR-16-1. Forpurposes of distinguishing the portion of a polycistronic pri-miRNAtranscript surrounding a given mature miRNA sequence, the pri-miRNA isherein referred as pri-miR-X, wherein “X” is the name of the maturemiRNA. For example, pri-miR-16-1 is the region of the polycistronicpri-miRNA transcript containing mature miR16-1. Alternatively, thepri-miRNA is referred to as “miR-X pri-miRNA”.

Once the Drosha recognition region in a pri-miRNA has been identified,one of skill in the art could identify small molecules capable ofbinding to the Drosha recognition region of a pri-miRNA. Methods foridentifying such small molecules are well known in the art, and involvethe use of mass spectrometry to identify small molecule compoundscapable of binding to structured RNA (e.g. U.S. Pat. Nos. 6,787,315;6,770,486; 6,730,485; 6,656,690, each of which is herein incorporated byreference in its entirety).

The present invention also provides oligomeric compounds useful in, forexample, the modulation of expression, endogenous levels, processing orfunction of small non-coding RNAs, in particular, pri-miRNAs.

As used herein, the term “small non-coding RNA” is used to encompass,without limitation, a polynucleotide molecule ranging from about 17 toabout 450 nucleotides in length, which can be endogenously transcribedor produced exogenously (chemically or synthetically), but is nottranslated into a protein. Examples of small non-coding RNAs include,but are not limited to, primary miRNA transcripts (also known aspri-pre-miRNAs, pri-mirs, pri-miRs and pri-miRNAs, which range fromaround 70 nucleotides to about 450 nucleotides in length and oftentaking the form of a hairpin structure); pre-miRNAs (also known aspre-mirs, pre-miRs and foldback miRNA precursors, which range fromaround 50 nucleotides to around 110 nucleotides in length); miRNAs (alsoknown as microRNAs, Mirs, miRs, mirs, and mature miRNAs, and generallyrefer either to double-stranded intermediate molecules around 17 toabout 25 nucleotides in length, or to single-stranded miRNAs, which maycomprise a bulged structure upon hybridization with a partiallycomplementary target nucleic acid molecule); or mimics of pri-miRNAs,pre-miRNAs or miRNAs. Small non-coding RNAs can be endogenouslytranscribed in cells, or can be synthetic oligonucleotides, in vitrotranscribed polynucleotides or nucleic acid oligomeric compoundsexpressed from vectors. Pri-miRNAs and pre-miRNAs, or mimics thereof,may be processed into smaller molecules. Preferred small non-coding RNAsof this invention are pri-miRNAs.

Small non-coding RNAs may include isolated single-, double-, ormultiple-stranded molecules, any of which may include regions ofintrastrand nucleobase complementarity, said regions capable of foldingand forming a molecule with fully or partially double-stranded ormultiple-stranded character based on regions of perfect or imperfectcomplementarity.

Oligomeric compounds of the invention modulate the levels, expression orfunction of small non-coding RNAs, particularly those encoded withinpolycistronic pri-miR transcripts, by hybridizing to a nucleic acidcomprising or encoding a small non-coding RNA nucleic acid targetresulting in alteration of normal function by, for example, facilitatingdestruction of the small non-coding RNA through cleavage, bysequestration, or by sterically occluding the function of the smallnon-coding RNA. These oligomeric compounds may be modified to increasedesired characteristics of the compounds, these modifications include,but are not limited to those which provide, improved pharmacokinetic orpharmacodynamic properties, binding affinity, stability, charge,localization or uptake.

As used herein, the terms “target nucleic acid,” “target RNA,” “targetRNA transcript” or “nucleic acid target” are used to encompass anynucleic acid capable of being targeted including, without limitation,RNA. In a one embodiment, the nucleic acids are non-coding sequencesincluding, but not limited to, pri-miRNAs (both polycistronic andmonocistronic pri-miRNAs), pre-miRNAs, and mature miRNAs. In anotherembodiment, the nucleic acid targets are single- or double-stranded, orsingle-stranded with partial double-stranded character; may occurnaturally within introns or untranslated regions of genes; and can beendogenously transcribed or exogenously produced.

In the context of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a small non-coding RNA, nucleicacid target, an RNA or protein associated with a small non-coding RNA,or a downstream target of the small non-coding RNA (e.g., a mRNArepresenting a protein-coding nucleic acid that is regulated by a smallnon-coding RNA). Inhibition is a suitable form of modulation and smallnon-coding RNA is a suitable target nucleic acid. Small non-coding RNAswhose levels can be modulated include pri-miRNAs (both polycistronic andmonocistronic pri-miRNAs), pre-miRNAs, and miRNAs.

In the context of the present invention, “modulation of function” meansan alteration in the function of the small non-coding RNA or analteration in the function of any cellular component with which thesmall non-coding RNA has an association or downstream effect.

The present invention provides, inter alia, oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications that render the compound capable ofsupporting modulation of the levels, expression or function of the smallnon-coding RNA by a degradation or cleavage mechanism.

The present invention also provides oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications that render the compound capable ofblocking or interfering with the levels, expression or function of oneor more small non-coding RNAs by steric occlusion.

The present invention also provides oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications or structural elements or motifs thatrender the compound capable of mimicking or replacing one or more smallnon-coding RNAs.

Oligomeric Compounds

In the context of the present invention, the term “oligomericcompound(s)” refers to polymeric structures which are capable ofhybridizing to at least a region of a small non-coding RNA molecule or atarget of small non-coding RNAs, or polymeric structures which arecapable of mimicking small non-coding RNAs. The term “oligomericcompound” includes, but is not limited to, compounds, comprisingoligonucleotides, oligonucleotides, oligonucleotide analogs,oligonucleotide mimetics and combinations of these. Oligomeric compoundsalso include, but are not limited to, antisense oligomeric compounds,antisense oligonucleotides, siRNAs, alternate splicers, primers, probesand other compounds that hybridize to at least a portion of the targetnucleic acid. Oligomeric compounds are routinely prepared linearly butcan be joined or otherwise prepared to be circular and may also includebranching. Separate oligomeric compounds can hybridize to form doublestranded compounds that can be blunt-ended or may include overhangs onone or both termini. In general, an oligomeric compound comprises abackbone of linked monomeric subunits where each linked monomericsubunit is directly or indirectly attached to a heterocyclic basemoiety. The linkages joining the monomeric subunits, the sugar moietiesor sugar surrogates and the heterocyclic base moieties can beindependently modified giving rise to a plurality of motifs for theresulting oligomeric compounds including hemimers, gapmers and chimeras.Modified oligomeric compounds are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. As used herein, the term“modification” includes substitution and/or any change from a startingor natural oligomeric compound, such as an oligonucleotide.Modifications to oligomeric compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or base moieties, such asthose described below.

The oligomeric compounds in accordance with this invention comprise fromabout 8 to about 80 monomeric subunits (i.e. from about 8 to about 80linked nucleosides). One of ordinary skill in the art will appreciatethat the invention embodies oligomeric compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits inlength, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 12 to50 monomeric subunits in length, as exemplified above.

In one embodiment, the oligomeric compounds of the invention are 13 to80 monomeric subunits in length, as exemplified above.

In one embodiment, the oligomeric compounds of the invention are 15 to30 monomeric subunits in length, as exemplified above.

In one embodiment, the oligomeric compounds of the invention are 17 to25 subunits in length, as exemplified herein.

As used herein, the term “about” means ±5% of the variable thereafter.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases that pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound of the invention is “specifically hybridizable”when association of the compound with the target nucleic acid interfereswith the normal function of the target nucleic acid to alter theactivity, disrupt the function, or modulate the level of the targetnucleic acid, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligomeric compound to non-targetnucleic acid sequences under conditions in which specific hybridizationis desired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under standard assay conditions inthe case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention; “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated. One having ordinary skill in the art willunderstand variability in the experimental protocols and be able todetermine when conditions are optimal for stringent hybridization withminimal non-specific hybridization events.

“Complementary,” as used herein, refers to the capacity for precisepairing of two monomeric subunits regardless of where in the oligomericcompound or target nucleic acid the two are located. For example, if amonomeric subunit at a certain position of an oligomeric compound iscapable of hydrogen bonding with a monomeric subunit at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligomeric compound and the target nucleic acid isconsidered to be a complementary position. The oligomeric compound andthe target nucleic acid are “substantially complementary” to each otherwhen a sufficient number of complementary positions in each molecule areoccupied by monomeric subunits that can hydrogen bond with each other.Thus, the term “substantially complementary” is used to indicate asufficient degree of precise pairing over a sufficient number ofmonomeric subunits such that stable and specific binding occurs betweenthe oligomeric compound and a target nucleic acid. The terms“substantially complementary” and “sufficiently complementary” areherein used interchangeably.

Generally, an oligomeric compound is “antisense” to a target nucleicacid when, written in the 5′ to 3′ direction, it comprises the reversecomplement of the corresponding region of the target nucleic acid.“Antisense compounds” are also often defined in the art to comprise thefurther limitation of, once hybridized to a target, being able to induceor trigger a reduction in target gene expression or target nucleic acidlevels.

It is understood in the art that the sequence of the oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization (e.g., a bulge, a loopstructure or a hairpin structure).

In some embodiments of the invention, the oligomeric compounds compriseat least 50%, at least 60%, at least 70%, at least 75%, at least 80%, orat least 85% sequence complementarity to a target region within thetarget nucleic acid. In other embodiments of the invention, theoligomeric compounds comprise at least 90% sequence complementarity to atarget region within the target nucleic acid. In other embodiments ofthe invention, the oligomeric compounds comprise at least 95% or atleast 99% sequence complementarity to a target region within the targetnucleic acid. For example, an oligomeric compound in which 18 of 20nucleobases of the oligomeric compound are complementary to a targetsequence would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an oligomericcompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anoligomeric compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

In some embodiments of the invention, the oligomeric compounds act asmimics or replacements for small non-coding RNAs. In this case, theoligomeric compounds of the invention can comprise at least 70% sequenceidentity to a small non-coding RNA or a region thereof. In someembodiments the oligomeric compounds of the invention can comprise atleast 90% sequence identity and in some embodiments can comprise atleast 95% sequence identity to to a small non-coding RNA or a regionthereof.

Oligomeric compounds, or portions thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific ISIS number.This identity may be over the entire length of the oligomeric compound,or in a portion of the oligomeric compound (e.g., nucleobases 1-20 of a27-mer may be compared to a 20-mer to determine percent identity of theoligomeric compound to the SEQ ID NO.) It is understood by those skilledin the art that an oligonucleotide need not have an identical sequenceto those described herein to function similarly to the oligonucleotidesdescribed herein. Shortened (i.e., deleted, and therefore non-identical)versions of oligonucleotides taught herein, or non-identical (i.e., onebase replaced with another) versions of the oligonucleotides taughtherein fall within the scope of the invention. Percent identity iscalculated according to the number of bases that are identical to theSEQ ID NO or compound to which it is being compared. The non-identicalbases may be adjacent to each other, dispersed through out theoligonucleotide, or both.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal sequence present in a portion of the modified sequence.Therefore, a 30 nucleobase oligonucleotide comprising the full sequenceof a 20 nucleobase SEQ ID NO would have a portion of 100% identity withthe 20 nucleobase SEQ ID NO while further comprising an additional 10nucleobase portion. In the context of the invention, the full length ofthe modified sequence may constitute a single portion.

“Targeting” an oligomeric compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose levels, expression or function is to be modulated. Thistarget nucleic acid may be, for example, a small non-coding RNA or itsprecursor (including a pri-miRNA), or a nucleic acid molecule from aninfectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe interaction to occur such that the desired effect, e.g., modulationof levels, expression or function, will result. Within the context ofthe present invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable sequence,structure, function, or characteristic. Within regions of target nucleicacids are segments. “Segments” are defined as smaller or sub-portions ofregions within a target nucleic acid. “Sites,” as used in the presentinvention, are defined as specific positions within a target nucleicacid. The terms region, segment, and site can also be used to describean oligomeric compound of the invention such as for example a gappedoligomeric compound having three separate segments.

Target regions include, but are not limited to, the following regions ofa pri-miRNA: the mature miRNA, the Drosha recognition region, the Droshacleavage site, the stem region of a predicted hairpin, or the loopregion of a predicted hairpin. A pri-miRNA target region may becontained within a polycistronic pri-miRNA transcript or a monocistronicpri-miRNA transcript. An miRNA gene may be found as a solitarytranscript, or it may be found within a 5′ untranslated region (5′UTR),within in an intron, or within a 3′ untranslated region (3′UTR) of agene. It is understood that a miRNA transcript derived from a miRNA genetranscript (i.e. a transcript which does not encode for a translatedprotein), or an miRNA transcript derived from an miRNA gene found within5′UTR, a 3′UTR, or an intron, are suitable target regions.

As exemplified herein, non-coding RNA genes and their products,including polycistronic miRNA genes, polycistronic pri-miRNAs,monocistronic pri-miRNAs, pre-miRNAs, and miRNAs, are also suitabletargets of the compounds of the invention.

The locations on the target nucleic acid to which compounds andcompositions of the invention hybridize are herein referred to as“suitable target segments.” As used herein the term “suitable targetsegment” is defined as at least an 8-nucleobase portion of a targetregion to which an oligomeric compound is targeted. Suitable targetsegments additionally include 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-,17-, 18, 19, 20, or 21 nucleobase portions of a target region to whichan oligomeric compound is targeted.

Once one or more targets, target regions, segments or sites have beenidentified, oligomeric compounds are designed to be sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect. The desired effectmay include, but is not limited to modulation of the levels, expressionor function of the target.

In some embodiments of the invention, the oligomeric compounds aredesigned to exert their modulatory effects via mimicking or targetingsmall non-coding RNAs associated with cellular factors that affect geneexpression, more specifically those involved in RNA or DNAmodifications. These modifications include, but are not limited to,posttranscriptional or chromosomal modifications such as methylation,acetylation, pseudouridylation or amination.

The oligomeric compounds of the invention may be in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor proteins to effect modulation of the levels, expression or functionof the target nucleic acid.

One non-limiting example of such a protein is the Drosha RNase IIIenzyme. Drosha is a nuclear enzyme that processes long primary RNAtranscripts (pri-miRNAs) from approximately 70 to 450 nucleotides inlength into pre-miRNAs (from about 50 to about 80 nucleotides in length)which are exported from the nucleus to encounter the human Dicer enzymewhich then processes pre-miRNAs into miRNAs. It is believed that, inprocessing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts thepri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′overhang(Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotideoverhang structure, a signature of RNaseIII enzymatic cleavage, has beenidentified as a critical specificity determinant in targeting andmaintaining small RNAs in the RNA interference pathway (Murchison, etal., Curr. Opin. Cell Biol., 2004, 16, 223-9).

A further non-limiting example involves the enzymes of the RISC complex.Use of the RISC complex to effect cleavage of RNA targets therebygreatly enhances the efficiency of oligonucleotide-mediated inhibitionof gene expression. Similar roles have been postulated for otherribonucleases such as those in the RNase III and ribonuclease L familyof enzymes.

Oligonucleotide Synthesis

Oligomeric compounds and phosphoramidites are made by methods well knownto those skilled in the art. Oligomerization of modified and unmodifiednucleosides is performed according to literature procedures for DNA likecompounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal(1993), Humana Press) and/or RNA like compounds (Scaringe, Methods(2001), 23, 206-217. Gait et al., Applications of Chemically synthesizedRNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al.,Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. Inaddition, specific protocols for the synthesis of oligomeric compoundsof the invention are illustrated in the examples below.

RNA oligomers can be synthesized by methods disclosed herein orpurchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.).

Irrespective of the particular protocol used, the oligomeric compoundsused in accordance with this invention may be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed.

Methods of isolation and analysis of oligonucleotides are well known inthe art. A 96-well plate format is particularly useful for thesynthesis, isolation and analysis of oligonucleotides.

RNA Synthesis

Methods of RNA synthesis are well known in the art (Scaringe, S. A.Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J.Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers,M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. andCaruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., etal., Acta Chem. Scand, 1990, 44, 639-641; Reddy, M. P., et al.,Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., NucleicAcids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron,1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,2315-2331).

Oligonucleotide Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. The respective ends of this linear polymeric structure can bejoined to form a circular structure by hybridization or by formation ofa covalent bond. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded structure. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside linkages of the oligonucleotide. Thenormal internucleoside linkage of RNA and DNA is a 3′ to 5′phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refersgenerally to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA). This term includes oligonucleotidescomposed of naturally occurring nucleobases, sugars and covalentinternucleoside linkages. The term “oligonucleotide analog” refers tooligonucleotides that have one or more non-naturally occurring portionswhich function in a similar manner to oligonucleotides. Suchnon-naturally occurring oligonucleotides are often selected overnaturally occurring forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for otheroligonucleotides or nucleic acid targets and increased stability in thepresence of nucleases.

In the context of this invention, the term “oligonucleoside” refers tonucleosides that are joined by internucleoside linkages that do not havephosphorus atoms. Internucleoside linkages of this type include shortchain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatomcycloalkyl, one or more short chain heteroatomic and one or more shortchain heterocyclic. These internucleoside linkages include but are notlimited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl,thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl,sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide,amide and others having mixed N, O, S and CH₂ component parts. Inaddition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodifications.

Modified Internucleoside Linkages

Specific examples of oligomeric compounds useful in this inventioninclude oligonucleotides containing modified, i.e. non-naturallyoccurring internucleoside linkages. Such non-naturally internucleosidelinkages are often selected over naturally occurring forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for other oligonucleotides or nucleic acid targets andincreased stability in the presence of nucleases. Oligomeric compoundsof the invention can have one or more modified internucleoside linkages.As defined in this specification, oligonucleotides having modifiedinternucleoside linkages include internucleoside linkages that retain aphosphorus atom and internucleoside linkages that do not have aphosphorus atom. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

A suitable phosphorus-containing modified internucleoside linkage is thephosphorothioate internucleoside linkage. Additional modifiedoligonucleotide backbones (internucleoside linkages) containing aphosphorus atom therein include, for example, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, each of which is herein incorporated by reference.

In other embodiments of the invention, oligomeric compounds have one ormore phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Modified oligonucleotide backbones (internucleoside linkages) that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA oligomeric compounds, thesugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA oligomeric compounds include,but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and5,719,262, each of which is herein incorporated by reference. Teachingof PNA oligomeric compounds can be found in Nielsen et al., Science,1991, 254, 1497-1500. PNA has been modified to incorporate numerousmodifications since the basic PNA structure was first prepared.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. A suitable class of linking groups have been selected togive a non-ionic oligomeric compound. The non-ionic morpholino-basedoligomeric compounds are less likely to have undesired interactions withcellular proteins. Morpholino-based oligomeric compounds are non-ionicmimics of oligonucleotides which are less likely to form undesiredinteractions with cellular proteins (Dwaine A. Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issuedJul. 23, 1991. The morpholino class of oligomeric compounds have beenprepared having a variety of different linking groups joining themonomeric subunits.

Another class of oligonucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in an DNA/RNAmolecule is replaced with a cyclohenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation. Furthermore the incorporation of CeNA into asequence targeting RNA was stable to serum and able to activate E. coliRNase resulting in cleavage of the target RNA strand.

Modified Sugar Moieties

Oligomeric compounds of the invention may also contain one or moremodified or substituted sugar moieties. The base units are maintainedfor hybridization with an appropriate nucleic acid target compound.Sugar modifications may impart nuclease stability, binding affinity orsome other beneficial biological property to the oligomeric compounds.Representative modified sugars include carbocyclic or acyclic sugars,sugars having substituent groups at one or more of their 2′, 3′ or 4′positions, sugars having substituents in place of one or more hydrogenatoms of the sugar, and sugars having a linkage between any two otheratoms in the sugar. These oligomeric compounds comprise a sugarsubstituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from1 to about 10. Some oligonucleotides comprise a sugar substituent groupselected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Onemodification that imparts increased nuclease resistance and a very highbinding affinity to nucleotides is the 2-methoxyethoxy (2′-MOE,2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997, 272,11944-12000). One of the immediate advantages of the 2′-MOE substitutionis the improvement in binding affinity, which is greater than manysimilar 2′ modifications such as O-methyl, O-propyl, and O-aminopropyl.Oligonucleotides having the 2′-O-methoxyethyl substituent also have beenshown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotideshaving the 2′-MOE modification displayed improved RNA affinity andhigher nuclease resistance. Oligomeric compounds having 2′-MOEmodifications are capable of inhibiting miRNA activity in vitro and invivo (Esau et al., J. Biol. Chem., 2004, 279, 52361-52365; U.S.Application Publication No. 2005/0261218).

Additional modifications include 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other sugar substituent groups include methoxy (—O—CH₃), aminopropoxy(—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) andfluoro (F). 2′-Sugar substituent groups may be in the arabino (up)position or ribo (down) position. One 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligomeric compounds may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of whichis herein incorporated by reference in its entirety.

Representative substituents groups are disclosed in U.S. Pat. No.6,172,209 entitled “Capped 2′-Oxyethoxy Oligonucleotides,” herebyincorporated by reference in its entirety.

Representative cyclic substituent groups are disclosed in U.S. Pat. No.6,271,358 entitled “RNA Targeted 2′-Oligomeric compounds that areConformationally Preorganized,” hereby incorporated by reference in itsentirety.

Particular sugar substituent groups include O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃))₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups are disclosed in U.S. Pat.No. 6,593,466 entitled “Functionalized Oligomers,” hereby incorporatedby reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Another group of modifications includes nucleosides having sugarmoieties that are bicyclic thereby locking the sugar conformationalgeometry. Such modifications may impart nuclease stability, bindingaffinity or some other beneficial biological property to the oligomericcompounds. The most studied of these nucleosides is a bicyclic sugarmoiety having a 4′-CH₂—O-2′ bridge. As can be seen in the structurebelow the 2′-O— has been linked via a methylene group to the 4′ carbon.This bridge attaches under the sugar as shown forcing the sugar ringinto a locked 3′-endo conformation geometry. The alpha-L nucleoside hasalso been reported wherein the linkage is above the ring and theheterocyclic base is in the alpha rather than the beta-conformation (seeU.S. Patent Application Publication No.: Application 2003/0087230). Thexylo analog has also been prepared (see U.S. Patent ApplicationPublication No.: 2003/0082807). The preferred bridge for a lockednucleic acid (LNA) is 4′-(—CH₂—)_(n)—O-2′ wherein n is 1 or 2. Theliterature is confusing when the term locked nucleic acid is used but ingeneral locked nucleic acids refers to n=1, ENA™ refers to n=2 (Kanekoet al., U.S. Patent Application Publication No.: US 2002/0147332, Singhet al., Chem. Commun., 1998, 4, 455-456, also see U.S. Pat. Nos.6,268,490 and 6,670,461 and U.S. Patent Application Publication No.: US2003/0207841). However the term locked nucleic acids can also be used ina more general sense to describe any bicyclic sugar moiety that has alocked conformation.

ENA™ along with LNA (n=1) have been studied more than the myriad ofother analogs. Oligomeric compounds incorporating LNA and ENA analogsdisplay very high duplex thermal stabilities with complementary DNA andRNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradationand good solubility properties.

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes; constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also beenprepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Some oligonucleotide mimetics have been prepared to include bicyclic andtricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta,1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121,3249-3255; and Renneberg et al., J. Am. Chem. Soc., 2002, 124,5993-6002). These modified nucleoside analogs have been oligomerizedusing the phosphoramidite approach and the resulting oligomericcompounds containing tricyclic nucleoside analogs have shown increasedthermal stabilities (Tm's) when hybridized to DNA, RNA and itself.Oligomeric compounds containing bicyclic nucleoside analogs have shownthermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acid and incorporates a phosphorus group inthe backbone. This class of olignucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Nucleobase Modifications

Oligomeric compounds of the invention may also contain one or morenucleobase (often referred to in the art simply as “base”) modificationsor substitutions which are structurally distinguishable from, yetfunctionally interchangeable with, naturally occurring or syntheticunmodified nucleobases. Such nucleobase modifications may impartnuclease stability, binding affinity or some other beneficial biologicalproperty to the oligomeric compounds. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases also referred to herein as heterocyclic basemoieties include other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Somenucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine.

In one aspect of the present invention oligomeric compounds are preparedhaving polycyclic heterocyclic compounds in place of one or moreheterocyclic base moieties. A number of tricyclic heterocyclic compoundshave been previously reported. These compounds are routinely used inantisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs.

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=H) (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀=S, R₁₁-R₁₄=H), (Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388). When incorporated into oligonucleotides, these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.Patent Application Publication 20030207804 and U.S. Patent ApplicationPublication 20030175906, both of which are incorporated herein byreference in their entirety).

Helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀=O, R₁₁=—O—(CH₂)₂—NH₂, R₁₂₋₁₄=H)(Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532).Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification. On the other hand, the gain in helical stabilitydoes not compromise the specificity of the oligonucleotides. The T_(m)data indicate an even greater discrimination between the perfect matchand mismatched sequences compared to dC5^(me). It was suggested that thetethered amino group serves as an additional hydrogen bond donor tointeract with the Hoogsteen face, namely the O6, of a complementaryguanine thereby forming 4 hydrogen bonds. This means that the increasedaffinity of G-clamp is mediated by the combination of extended basestacking and additional specific hydrogen bonding.

Tricyclic heterocyclic compounds and methods of using them that areamenable to the present invention are disclosed in U.S. Pat. No.6,028,183, and U.S. Pat. No. 6,007,992, the contents of both areincorporated herein in their entirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their sequence specificity makes them valuable nucleobase analogsfor the development of more potent antisense-based drugs. In fact,promising data have been derived from in vitro experiments demonstratingthat heptanucleotides containing phenoxazine substitutions can activateRNaseH, enhance cellular uptake and exhibit an increased antisenseactivity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532). The activity enhancement was even more pronounced in case ofG-clamp, as a single substitution was shown to significantly improve thein vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides(Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner,R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).

Modified polycyclic heterocyclic compounds useful as heterocyclic basesare disclosed in but not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653;5,763,588; 6,005,096; and 5,681,941, and U.S. Patent ApplicationPublication 20030158403, each of which is incorporated herein byreference in its entirety.

Certain nucleobase substitutions, including 5-methylcytosinesubstitutions, are particularly useful for increasing the bindingaffinity of the oligonucleotides of the invention. For example,5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Conjugated Oligomeric Compounds

One substitution that can be appended to the oligomeric compounds of theinvention involves the linkage of one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe resulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, carbohydrates, lipids, phospholipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve oligomer uptake, enhance oligomer resistance todegradation, and/or strengthen hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Furthermore, the oligomeric compounds of the invention can have one ormore moieties bound or conjugated, which facilitates the active orpassive transport, localization, or compartmentalization of theoligomeric compound. Cellular localization includes, but is not limitedto, localization to within the nucleus, the nucleolus, or the cytoplasm.Compartmentalization includes, but is not limited to, any directedmovement of the oligonucleotides of the invention to a cellularcompartment including the nucleus, nucleolus, mitochondrion, orimbedding into a cellular membrane. Furthermore, the oligomericcompounds of the invention comprise one or more conjugate moieties whichfacilitate posttranscriptional modification.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example: aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example Wincott et al., WO97/26270, incorporated by reference herein). These terminalmodifications protect the oligomeric compounds having terminal nucleicacid molecules from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present onboth termini. For double-stranded oligomeric compounds, the cap may bepresent at either or both termini of either strand. In non-limitingexamples, the 5′-cap includes inverted abasic residue (moiety),4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety (see Wincott et al., International PCTpublication No. WO 97/26270, incorporated by reference herein).

Particularly preferred 3′-cap structures of the present inventioninclude, for example 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

Synthesis of Chimeric Oligonucleotides

In preferred embodiments, oligomeric compounds of the invention are“uniformly modified”, i.e., each nucleotide position bears anon-naturally occurring internucleoside linkage, sugar moiety and/ornucleobase. The present invention further encompasses “positionallymodified” oligomeric compounds, in which one or more of theaforementioned modifications is incorporated in a single oligomericcompound (e.g. each nucleotide within an oligonucleotide may contain adifferent modification, the same modification, or be unmodified) or evenat a single monomeric subunit such as a nucleoside (e.g. a nucleosidemay contain both a sugar modification and a base modification) within aoligomeric compound. In one non-limiting example, a preferred oligomericcompound is modified such that each sugar moiety is a 2′-MOE nucleotide,each internucleoside linkage is a phosphorothioate linkage, and eachcytosine is a 5-methyl cytosine. The present invention also includesoligomeric compounds which are chimeric oligomeric compounds. “Chimeric”oligomeric compounds or “chimeras,” in the context of this invention,are oligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, an oligomeric compound may be designed tocomprise a region that serves as a substrate for RNase H. RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H by an oligomeric compound having a cleavageregion, therefore, results in cleavage of the RNA target, therebyenhancing the efficiency of the oligomeric compound. Consequently,comparable results can often be obtained with shorter oligomericcompounds having substrate regions when chimeras are used, compared tofor example phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotidemimics, oligonucleotide analogs, oligonucleosides and/or oligonucleotidemimetics as described above. Such oligomeric compounds have also beenreferred to in the art as hybrids, hemimers, gapmers or invertedgapmers. Representative U.S. patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference in its entirety.

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers.” Methods ofsynthesizing chimeric oligonucleotides are well known in the art.

Nucleotides, both native and modified, have a certain conformationalgeometry which affects their hybridization and affinity properties. Theterms used to describe the conformational geometry of homoduplex nucleicacids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as, but notlimited to, antisense mechanisms, including RNase H-mediated and RNAinterference mechanisms, as these mechanisms involved the hybridizationof a synthetic sequence strand to an RNA target strand. In the case ofRNase H, effective inhibition of the mRNA requires that the antisensesequence achieve at least a threshold of hybridization.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependent on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is alsocorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in FIG. 2, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation.

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA-like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but are notlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric compounds designed to act as triggers ofRNAi having one or more nucleosides modified in such a way as to favor aC3′-endo type conformation. Along similar lines, oligomeric triggers ofRNAi response might be composed of one or more nucleosides modified insuch a way that conformation is locked into a C3′-endo typeconformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun.(1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita etal, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)

The conformation of modified nucleosides and their oligomers can beestimated by various methods such as molecular dynamics calculations,nuclear magnetic resonance spectroscopy and CD measurements. Hence,modifications predicted to induce RNA-like conformations (A-form duplexgeometry in an oligomeric context), are useful in the oligomericcompounds of the present invention. The synthesis of modifiednucleosides amenable to the present invention are known in the art (seefor example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. LeroyB. Townsend, 1988, Plenum Press.)

In one aspect, the present invention is directed to oligomeric compoundsthat are designed to have enhanced properties compared to native RNA.One method to design optimized or enhanced oligomeric compounds involveseach nucleoside of the selected sequence being scrutinized for possibleenhancing modifications. One modification would be the replacement ofone or more RNA nucleosides with nucleosides that have the same 3′-endoconformational geometry. Such modifications can enhance chemical andnuclease stability relative to native RNA while at the same time beingmuch cheaper and easier to synthesize and/or incorporate into anoligonucleotide. The sequence can be further divided into regions andthe nucleosides of each region evaluated for enhancing modificationsthat can be the result of a chimeric configuration. Consideration isalso given to the 5′ and 3′-termini as there are often advantageousmodifications that can be made to one or more of the terminalnucleosides. The oligomeric compounds of the present invention mayinclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double-stranded sequence or sequences.Other modifications considered are internucleoside linkages, conjugategroups, substitute sugars or bases, substitution of one or morenucleosides with nucleoside mimetics and any other modification that canenhance the desired property of the oligomeric compound.

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆,straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, C₁-C₈, orC₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbylcontaining at least one, or about 1 to about 3 hetero atoms in thechain, including the terminal portion of the chain. Suitable heteroatomsinclude N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, orC₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkenyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkynyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon. Thenumber of carbon atoms can vary from 1 to about 12, from 1 to about 6,and the total number of ring members varies from three to about 15, orfrom about 3 to about 8. Suitable ring heteroatoms are N, O and S.Suitable heterocycloalkyl groups include, but are not limited to,morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl,homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl,tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl,tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, andtetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Suitable aryl rings haveabout 6 to about 20 ring carbons. Especially suitable aryl rings includephenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. The ring system can contain about 1 to about 4 rings.The number of carbon atoms can vary from 1 to about 12, from 1 to about6, and the total number of ring members varies from three to about 15,or from about 3 to about 8. Suitable ring heteroatoms are N, O and S.Suitable hetaryl moieties include, but are not limited to, pyrazolyl,thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl,purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl,etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Suitable halo (halogen) substituents are Cl, Br, andI.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc. In all the preceding formulae, the squiggle (˜) indicatesa bond to an oxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. No.5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat.No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S.Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expresslyincorporated herein by reference in its entirety.

Screening Oligomeric Compounds

Screening methods for the identification of effective modulators ofsmall non-coding RNAs, including pri-miRNAs, are also comprehended bythe instant invention and comprise the steps of contacting a smallnon-coding RNA, or portion thereof, with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the levels, expression or alter the function of thesmall non-coding RNA. As described herein, the candidate modulator canbe an oligomeric compound targeted to a pri-miRNA, or any portionthereof, including the mature miRNA, the Drosha recognition region, theDrosha cleavage region, the stem of the hairpin, or the loop of thehairpin. Candidate modulators further include small molecule compoundsthat bind to structured regions of small non-coding RNAs, such asstructured regions within pri-miRNAs. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the levels, expression or altering thefunction of the small non-coding RNA, the modulator may then be employedin further investigative studies, or for use as a target validation,research, diagnostic, or therapeutic agent in accordance with thepresent invention. In one embodiment, the candidate modulator isscreened for its ability to cause the accumulation of pri-miRNA levels.

As described herein, oligomeric compounds are further used to identifyDrosha recognition regions.

Screening methods for the identification of small non-coding RNA mimicsare also within the scope of the invention. Screening for smallnon-coding RNA modulators or mimics can also be performed in vitro, exvivo, or in vivo by contacting samples, tissues, cells or organisms withcandidate modulators or mimics and selecting for one or more candidatemodulators which show modulatory effects.

Design and Screening of Duplexed Oligomeric Compounds

In screening and target validation studies, oligomeric compounds of theinvention can be used in combination with their respective complementarystrand oligomeric compound to form stabilized double-stranded (duplexed)oligonucleotides. In accordance with the present invention, a series ofduplexes comprising the oligomeric compounds of the present inventionand their complements can be designed to target a small non-coding RNA.The ends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in some embodiments, both strands of theduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini, as described supra.

In some embodiments, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 1) may be prepared with bluntends (no single stranded overhang) as shown:

In other embodiments, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 1), having a two-nucleobaseoverhang of deoxythymidine (dT) and its complement sense strand may beprepared with overhangs as shown:

These sequences are shown to contain thymine (T) but one of skill in theart will appreciate that thymine (T) is generally replaced by uracil (U)in RNA sequences. RNA strands of the duplex can be synthesized bymethods disclosed herein or purchased from Dharmacon Research Inc.(Lafayette, Colo.).

Diagnostics, Drug Discovery and Therapeutics

The oligomeric compounds and compositions of the present invention canadditionally be utilized for research, drug discovery, kits anddiagnostics, and therapeutics.

For use in research, oligomeric compounds of the present invention areused to interfere with the normal function of the nucleic acid moleculesto which they are targeted. Expression patterns within cells or tissuestreated with one or more oligomeric compounds or compositions of theinvention are compared to control cells or tissues not treated with thecompounds or compositions and the patterns produced are analyzed fordifferential levels of nucleic acid expression as they pertain, forexample, to disease association, signaling pathway, cellularlocalization, expression level, size, structure or function of the genesexamined. These analyses can be performed on stimulated or unstimulatedcells and in the presence or absence of other compounds that affectexpression patterns.

For use in drug discovery, oligomeric compounds of the present inventionare used to elucidate relationships that exist between small non-codingRNAs, genes or proteins and a disease state, phenotype, or condition.These methods include detecting or modulating a target comprisingcontacting a sample, tissue, cell, or organism with the oligomericcompounds and compositions of the present invention, measuring thelevels of the target and/or the levels of downstream gene productsincluding mRNA or proteins encoded thereby, a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to an untreated sample, a positive control or anegative control. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa disease.

For use in kits and diagnostics, the oligomeric compounds andcompositions of the present invention, either alone or in combinationwith other compounds or therapeutics, can be used as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of non-coding or codingnucleic acids expressed within cells and tissues.

The specificity and sensitivity of compounds and compositions can alsobe harnessed by those of skill in the art for therapeutic uses.Antisense oligomeric compounds have been employed as therapeuticmoieties in the treatment of disease states in animals, includinghumans. Antisense oligonucleotide drugs, including ribozymes, have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that oligomericcompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder presenting conditions that can be treated,ameliorated, or improved by modulating the expression of a selectedsmall non-coding target nucleic acid is treated by administering thecompounds and compositions. For example, in one non-limiting embodiment,the methods comprise the step of administering to or contacting theanimal, an effective amount of a modulator or mimic to treat, ameliorateor improve the conditions associated with the disease or disorder. Thecompounds of the present invention effectively modulate the activity orfunction of the small non-coding RNA target or inhibit the expression orlevels of the small non-coding RNA target. In preferred embodiments, thesmall non-coding RNA target is a polycistronic pri-miRNA, amonocistronic pri-miRNA, a pre-miRNA, or a miRNA. In additionalembodiments, the small non-coding RNA target is a single member of amiRNA family. Alternatively, two or more members of an miRNA family areselected for modulation. In a further embodiment, the small non-codingRNA target is a selectively processed miRNA. In one embodiment, thelevel, activity or expression of the target in an animal is inhibited byabout 10%. In another embodiment the level, activity or expression of atarget in an animal is inhibited by about 30%. Further, the level,activity or expression of a target in an animal is inhibited by 50% ormore, by 60% or more, by 70% or more, by 80% or more, by 90% or more, orby 95% or more. In another embodiment, the present invention providesfor the use of a compound of the invention in the manufacture of amedicament for the treatment of any and all conditions associated withmiRNAs and miRNA families.

The reduction of target levels may be measured in serum, adipose tissue,liver or any other body fluid, tissue or organ of the animal known tocontain the small non-coding RNA or its precursor. Further, the cellscontained within the fluids, tissues or organs being analyzed contain anucleic acid molecule of a downstream target regulated or modulated bythe small non-coding RNA target itself.

Compositions and Methods for Formulating Pharmaceutical Compositions

The present invention also include pharmaceutical compositions andformulations that include the oligomeric compounds, small non-codingRNAs and compositions of the invention. Compositions and methods for theformulation of pharmaceutical compositions are dependent upon a numberof criteria, including, but not limited to, route of administration,extent of disease, or dose to be administered. Such considerations arewell understood by those skilled in the art.

The oligomeric compounds and compositions of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofthe compound or composition to a suitable pharmaceutically acceptablediluent or carrier. Use of the oligomeric compounds and methods of theinvention may also be useful prophylactically.

The oligomeric compounds and compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds and compositionsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. Suitable examples include, but are notlimited to, sodium and potassium salts.

In some embodiments, an oligomeric compound can be administered to asubject via an oral route of administration. The subject may be amammal, such as a mouse, a rat, a dog, a guinea pig, or a non-humanprimate. In some embodiments, the subject may be a human or a humanpatient. In certain embodiments, the subject may be in need ofmodulation of the level or expression of one or more pri-miRNAs asdiscussed in more detail herein. In some embodiments, compositions foradministration to a subject will comprise modified oligonucleotideshaving one or more modifications, as described herein.

Cell Culture and Oligonucleotide Treatment

The effects of oligomeric compounds on target nucleic acid expression orfunction can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can bereadily determined by methods routine in the art, for example Northernblot analysis, ribonuclease protection assays, or real-time PCR. Celltypes used for such analyses are available from commercial vendors (e.g.American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., ResearchTriangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cellsare cultured according to the vendor's instructions using commerciallyavailable reagents (e.g. Invitrogen Life Technologies, Carlsbad,Calif.). Illustrative cell types include, but are not limited to: T-24cells, A549 cells, normal human mammary epithelial cells (HMECs), MCF7cells, T47D cells, BJ cells, B16-F10 cells, human vascular endothelialcells (HUVECs), human neonatal dermal fibroblast (NHDF) cells, humanembryonic keratinocytes (HEK), 293T cells, HepG2, human preadipocytes,human differentiated adipocytes (preapidocytes differentiated accordingto methods known in the art), NT2 cells (also known as NTERA-2 cl.D1),and HeLa cells.

Treatment with Antisense Oligomeric Compounds

In general, when cells reach approximately 80% confluency, they aretreated with oligomeric compounds of the invention. Oligomeric compoundsare introduced into cells using the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Oligomeric compounds aremixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.) toachieve the desired final concentration of oligomeric compound andLIPOFECTIN®. Before adding to cells, the oligomeric compound,LIPOFECTIN® and OPTI-MEM® 1 are mixed thoroughly and incubated forapproximately 0.5 hrs. The medium is removed from the plates and theplates are tapped on sterile gauze. Each well of a 96-well plate iswashed with 150 μl of phosphate-buffered saline or Hank's balanced saltsolution. Each well of a 24-well plate is washed with 250 μL ofphosphate-buffered saline or Hank's balanced salt solution. The washbuffer in each well is replaced with 100 μL or 250 μL of the oligomericcompound/OPTI-MEM® 1/LIPOFECTIN® cocktail for 96-well or 24-well plates,respectively. Untreated control cells receive LIPOFECTIN® only. Theplates are incubated for approximately 4 to 7 hours at 37° C., afterwhich the medium is removed and the plates are tapped on sterile gauze.100 μl or 1 mL of full growth medium is added to each well of a 96-wellplate or a 24-well plate, respectively. Cells are harvested 16-24 hoursafter oligonucleotide treatment, at which time RNA can be isolated andtarget reduction measured by real-time PCR, or other phenotypic assaysperformed. In general, data from treated cells are obtained intriplicate, and results presented as an average of the three trials.

Alternatively, cells are transfected using LIPOFECTAMINE® (Invitrogen,Carlsbad, Calif.). When cells reached 65-75% confluency, they aretreated with oligonucleotide. Oligonucleotide is mixed withLIPOFECTAMINE® in OPTI-MEM® 1 reduced serum medium (Invitrogen,Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide and a LIPOFECTAMINE® concentration of ranging from 2 to12 μg/mL per 100 nM oligonucleotide. This transfection mixture isincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells are washed once with 100 μL OPTI-MEM® 1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture is replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

In some embodiments, cells are transiently transfected with oligomericcompounds of the instant invention. In some embodiments, cells aretransfected and selected for stable expression of an oligomeric compoundof the instant invention.

The concentration of oligonucleotide used varies from cell line to cellline. Methods to determine the optimal oligonucleotide concentration fora particular cell line are well known in the art. For example, the cellsare treated with a positive control oligonucleotide targeting a genesuch as H-ras, at a range of concentrations. Controls may be unmodified,uniformly modified, or chimeric oligomeric compounds. The concentrationof positive control oligonucleotide that results in, for example, 80%inhibition of the control target RNA is then be utilized as thescreening concentration for new oligonucleotides in subsequentexperiments for that cell line. If 80% inhibition is not achieved, thelowest concentration of positive control oligonucleotide that results in60% inhibition of target expression or function is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. The concentrations of oligonucleotides used herein canrange from 1 nM to 300 nM.

Analysis of Oligonucleotide Inhibition of a Target Levels or Expression

Modulation of target levels or expression can be assayed in a variety ofways known in the art. For example, target nucleic acid levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or quantitative real-time PCR. RNA analysis can beperformed on total cellular RNA or poly(A)+ mRNA. Methods of RNAisolation are well known in the art. Northern blot analysis is alsoroutine in the art. Quantitative real-time PCR can be convenientlyaccomplished using the commercially available ABI PRISM® 7600, 7700, or7900 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Additional examples of methods of gene expression analysis known in theart include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE(serial analysis of gene expression) (Madden, et al., Drug Discov.Today, 2000, 5, 415-425), READS (restriction enzyme amplification ofdigested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303,258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc.Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays andproteomics (Cells, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, etal., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST)sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al.,J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF)(Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al.,Cytometry, 2000, 41, 203-208), subtractive cloning, differential display(DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21),comparative genomic hybridization (Carulli, et al., J. Cell Biochem.Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization)techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904),and mass spectrometry methods (To, Comb. Chem. High Throughput Screen,2000, 3, 235-41).

RNA Isolation

RNA is prepared from cell lines such as HeLa, NT2, T-24, and A549 usingmethods well known in the art, for example, using the TRIZOL®(Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols. Briefly, cell monolayers are washed twice withcold PBS, and cells are lysed using TRIZOL® (Invitrogen, Carlsbad,Calif.) at a volume of 1 mL per 10 cm² culture dish surface area, andtotal RNA is prepared according to the TRIZOL® protocol.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels is accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mMMgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forwardprimer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor,1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution(20-200 ng). The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as GAPDH, or by quantifying total RNA using RIBOGREEN®(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN® working reagent (RIBOGREEN® reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CYTOFLUOR® 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers are designed to hybridize to the target sequence.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

Northern Blot Analysis of Target RNA Levels

Northern blot analysis is performed according to routine proceduresknown in the art. Fifteen to twenty micrograms of total RNA isfractionated by electrophoresis through 10% acrylamide urea gels using aTBE buffer system (Invitrogen). RNA is transferred from the gel toHYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway,N.J.) by electroblotting in an Xcell SURELOCK™ Mincell (Invitrogen,Carlsbad, Calif.). Membranes are fixed by UV cross-linking using aSTRATALINKER® UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using RAPID HYB™ buffer solution (Amersham) usingmanufacturer's recommendations for oligonucleotide probes.

A target specific DNA oligonucleotide probe with the sequence is used todetect the RNA of interest. Probes used to detect miRNAs are synthesizedby commercial vendors such as IDT (Coralville, Iowa). The probe is 5′end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega,Madison, Wis.). To normalize for variations in loading and transferefficiency membranes are stripped and re-probed for U6 RNA. Hybridizedmembranes are visualized and quantitated using a STORM® 860PHOSPHORIMAGER® System and IMAGEQUANT® Software V3.3 (MolecularDynamics, Sunnyvale, Calif.).

Analysis of Protein Levels

Protein levels of a downstream target modulated or regulated by a smallnon-coding RNA can be evaluated or quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA),quantitative protein assays, protein activity assays (for example,caspase activity assays), immunohistochemistry, immunocytochemistry orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Phenotypic Assays

Once modulators are designed or identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive orsuggestive of efficacy in the treatment, amelioration or improvement ofphysiologic conditions associated with a particular disease state orcondition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays include cell cycle assays, apoptosis assays,angiogenesis assays (e.g. endothelial tube formation assays, angiogenicgene expression assays, matrix metalloprotease activity assays),adipocyte assays (e.g. insulin signaling assays, adipocytedifferentiation assays), inflammation assays (e.g. cytokine signalingassays, dendritic cell cytokine production assays); examples of suchassays are readily found in the art (e.g., U.S. Application PublicationNo. 2005/0261218, which is hereby incorporated by reference in itsentirety). Additional phenotypic assays include those that evaluatedifferentiation and dedifferentiation of stem cells, for example, adultstem cells and embryonic stem cells; protocols for these assays are alsowell known in the art (e.g. Turksen, Embryonic Stem Cells: Methods andProtocols, 2001, Humana Press; Totowa, N.J.; Klug, Hematopoietic StemCell Protocols, 2001, Humana Press, Totowa, N.J.; Zigova, Neural StemCells: Methods and Protocols, 2002, Humana Press, Totowa, N.J.).

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, GENBANK®accession numbers, and the like) cited in the present application isspecifically incorporated herein by reference in its entirety.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner. Throughout theseexamples, molecular cloning reactions, and other standard recombinantDNA techniques, were carried out according to routine methods, such asthose described in Maniatis et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Cold Spring Harbor Press (1989), using commerciallyavailable reagents, except where otherwise noted.

EXAMPLES Example 1 Effects of Oligomeric Compounds on Expression ofpri-miRNAs

As described herein, pri-miRNAs, often hundreds of nucleotides inlength, are processed by a nuclear enzyme in the RNase III family knownas Drosha, into approximately 70 nucleotide-long pre-miRNAs (also knownas stem-loop structures, hairpins, pre-miRs or foldback miRNAprecursors), and pre-miRNAs are subsequently exported from the nucleusto the cytoplasm, where they are processed by human Dicer intodouble-stranded miRNAs, which are subsequently processed by the DicerRNase into mature miRNAs. It is believed that, in processing thepri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA atthe base of the mature miRNA, leaving a 2-nucleotide 3′overhang (Lee, etal., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhangstructure, a signature of RNaseIII cleavage, has been identified as acritical specificity determinant in targeting and maintaining small RNAsin the RNA interference pathway (Murchison, et al., Curr. Opin. CellBiol., 2004, 16, 223-9).

The oligomeric compounds of the present invention are believed todisrupt pri-miRNA and/or pre-miRNA structures, and sterically hinderDrosha and/or Dicer cleavage, respectively. Additionally, oligomericcompounds capable of binding to the mature miRNA are believed to preventthe RISC-mediated binding of a miRNA to its mRNA target, either bydegradation or steric occlusion of the miRNA.

The levels of pri-miR-15a were compared in HepG2 cells treated with aseries of chimeric oligomeric compounds, targeting and spanning theentire length of pri-miR-15a; these compounds are shown in Table 1,below. The compounds were designed using publicly available sequenceinformation (the complement of nucleotides 31603159 to 31603468 ofGENBANK® Accession number NT_(—)024524.13, deposited with GENBANK® onOct. 7, 2003). Each chimeric oligomeric compound is 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings.” These chimeric compounds havinga central gap region are herein referred to as “gapmers”. The wings arecomposed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Using thetransfection methods described herein, HepG2 cells were treated with 100nM of each of these gapmer oligomeric compounds. Total RNA was isolatedfrom HepG2 cells by lysing cells in 1 mL TRIZOL® (Invitrogen, Carlsbad,Calif.) using the manufacturer's recommended protocols. The isolated RNAwas used as the substrate in the reverse transcriptase, real-time PCRassays, as described herein. Real-time PCR analysis was performed usinga primer/probe set specific for pri-miR-15a to assess the effects ofthese compounds on levels of pri-miR-15a. ISIS 339317(GTGTGTTTAAAAAAAATAAAACCTTGGA; SEQ ID NO.: 6) was used as the forwardprimer, ISIS 339318 (TGGCCTGCACCTTTTCAAA; SEQ ID NO.: 7) was used as thereverse primer, and ISIS 339319 (AAAGTAGCAGCACATAATGGTTTGTGG; SEQ IDNO.: 8) was used as the probe. Total RNA was quantified using RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.),levels observed for each target are normalized to 5.8S rRNA, and valuesare expressed relative to the untreated control. Reduction in the levelsof pri-miR-15a by these gapmer oligomeric compounds is expressed as apercentage of RNA levels in untreated control cells. Results of theseexperiments are described in Table 1.

TABLE 1 Effects of chimeric oligomeric compoundson expression of pri-miR-15a Expression of ISIS SEQ ID pri-miR-15aNumber NO Sequence (% UTC) 347964 9 TATAACATTGATGTAATATG 13.7 347965 10GCTACTTTACTCCAAGGTTT 86.0 347966 11 TGCTACTTTACTCCAAGGTT 39.2 347967 12GCACCTTTTCAAAATCCACA 152.3 347968 13 CCTGCACCTTTTCAAAATCC 8.4 347969 14TGGCCTGCACCTTTTCAAAA 39.5 347970 15 ATATGGCCTGCACCTTTTCA 2.2 347971 16ACAATATGGCCTGCACCTTT 92.8 347972 17 AGCACAATATGGCCTGCACC 98.6 347973 18GGCAGCACAATATGGCCTGC 143.3 347974 19 TGAGGCAGCACAATATGGCC 98.1 347975 20TTTTGAGGCAGCACAATATG 9.2 347976 21 TATTTTTGAGGCAGCACAAT 73.0 347977 22TTGTATTTTTGAGGCAGCAC 111.3 347978 23 TCCTTGTATTTTTGAGGCAG 51.1 347979 24AGATCCTTGTATTTTTGAGG 74.9 347980 25 AGATCAGATCCTTGTATTTT 3.6 347981 26AGAAGATCAGATCCTTGTAT N/D 347982 27 TTCAGAAGATCAGATCCTTG 82.2 347983 28AAATATATTTTCTTCAGAAG 13.0

From these data, it was observed that oligomeric compounds ISIS 347964,347966, 347968, 347970, 347975, 347980 and 347983 show significantinhibition of levels of pri-miR-15a. Thus, it is possible that theantisense oligomeric compounds ISIS 347964, 347966, 347968, 347970,347975, 347980 and 347983 bind to pri-miR-15a and/or pre-miR-15amolecules and cause their degradation and cleavage.

From these data, it was observed that oligomeric compounds ISIS 347967,347977 and 347973 stimulated an increase of pri-miR-15a levels. It ispossible that the oligomeric compounds ISIS 347967, 347977 and 347973bind to pri-miR-15a and inhibit its processing into mature miR-15a.

In addition, uniform 2′-MOE and 2′-MOE gapmer oligomeric compoundstargeting mature miR-15a-1 and mature miR-15b were transfected into T47Dcells, for analysis of their effects on pri-miR-15a-1 and/or pri-miR-15blevels. The oligomeric compounds ISIS 327927 (SEQ ID NO: 29), a uniform2′-MOE compound, and ISIS 345391 (SEQ ID NO: 29), a 2′-MOE 5-10-7 gapmercompound, both target mature miR-15b. The oligomeric compounds ISIS327951 (SEQ ID NO: 30), a uniform 2′-MOE compound, and ISIS 345411 (SEQID NO: 30), a 2′-MOE 5-10-7 gapmer compound, both target maturemiR-15a-1. Oligomeric compounds ISIS 129686 (CGTTATTAACCTCCGTTGAA; SEQID NO: 31), ISIS 129691 (ATGCATACTACGAAAGGCCG; SEQ ID NO:32), and ISIS116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 33; targeting an unrelatedgene, PTEN), are not designed to target to mature miR-15b or maturemiR-15-a-1, and were used as negative controls. ISIS 129686, 129691, and116847 are phosphorothiated 2′-MOE 5-10-5 gapmers, and all cytosines are5-methylcytosines. T47D cells (seeded in 12-well plates) were treatedwith these oligomeric compounds, RNA was isolated from the treated cellsby lysing in 1 mL TRIZOL® (Invitrogen, Carlsbad, Calif.) and total RNAwas prepared using the manufacturer's recommended protocols. To assessthe effects of these compounds on pri-miR-15a and/or pri-miR-15b levels,real-time PCR analysis was performed using either the primer/probe setspecific for pri-miR-15a described above, or a primer probe set specificfor pri-miR-15b: ISIS 339320 (CCTACATTTTTGAGGCCTTAAAGTACTG; SEQ ID NO:34) was used as the forward primer for the pri-miR-15b, ISIS 339321(CAAATAATGATTCGCATCTTGACTGT; SEQ ID NO: 35) was used as the reverseprimer for the pri-miR-15b, and ISIS 339322 (AGCAGCACATCATGGTTTACATGC;SEQ ID NO: 36) was used as the probe. Total RNA was quantified usingRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.), levels observed for each target were normalized to 5.8S rRNA,and values are expressed relative to the untreated control. Inhibitionof pri-miR-15a or pri-miR-15b levels upon treatment with theseoligomeric compounds is was assessed and expressed as a percentage ofRNA levels in untreated control cells.

Following repeated experimentation, it was observed that the uniform2′-MOE oligomeric compounds ISIS 327927 (SEQ ID NO: 29) and ISIS 327951(SEQ ID NO: 30), targeted to the mature miR-15b and mature miR-15a-1,respectively, each stimulate an approximately 2.5- to 3.5-fold increasein level of pri-miR-15a and an approximately 1.5- to 2.5-fold increasein the level of pri-miR-15b. Therefore, it is possible that ISIS 327927and 327951 can bind to pri-miR-15a and pri-miR-15b, or their respectivepre-miRNAs, and interfere with their processing into the mature miR-15aor mature miR-15b.

In accordance with the present invention, a nested series of uniform2′-MOE oligomeric compounds were designed and synthesized to target theentire length of pri-miR-15a, using publicly available sequenceinformation (the complement of nucleotides 31603159 to 31603468 ofGENBANK® Accession number NT_(—)024524.13, deposited with GENBANK® onOct. 7, 2003). The compounds are shown in Table 2. Each compound is 19nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotidesand phosphorothioate (P═S) internucleoside linkages throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds are analyzed for their effect on mature miRNA, pre-miRNA orpri-miRNA levels by quantitative real-time PCR; alternatively, they areused in other assays to investigate the role of miRNAs or the functionof targets downstream of miRNAs.

TABLE 2 Uniform 2′-MOE PS Compounds targeting pri-miR-15a SEQ ID ISIS NOSequence 356213 9 TATAACATTGATGTAATATG 356214 10 GCTACTTTACTCCAAGGTTT356215 11 TGCTACTTTACTCCAAGGTT 356216 12 GCACCTTTTCAAAATCCACA 356217 13CCTGCACCTTTTCAAAATCC 356218 14 TGGCCTGCACCTTTTCAAAA 356219 15ATATGGCCTGCACCTTTTCA 356220 16 ACAATATGGCCTGCACCTTT 356221 17AGCACAATATGGCCTGCACC 356222 18 GGCAGCACAATATGGCCTGC 356223 19TGAGGCAGCACAATATGGCC 356224 20 TTTTGAGGCAGCACAATATG 356225 21TATTTTTGAGGCAGCACAAT 356226 22 TTGTATTTTTGAGGCAGCAC 356227 23TCCTTGTATTTTTGAGGCAG 356228 24 AGATCCTTGTATTTTTGAGG 356229 25AGATCAGATCCTTGTATTTT 356230 26 AGAAGATCAGATCCTTGTAT 356231 27TTCAGAAGATCAGATCCTTG 356232 28 AAATATATTTTCTTCAGAAG

Using the reverse transcriptase and real-time PCR methods described, thelevels of pri-miR-15a were compared in T47D cells treated with thenested series of uniform 2′-MOE oligomeric compounds, targeting andspanning the entire length of pri-miR-15a. ISIS 356215 (SEQ ID NO: 11)targets a region flanking and immediately 5′ to the predicted 5′ Droshacleavage site in pri-miR-15a. ISIS 356218 (SEQ ID NO: 14) targets aregion in the loop of pri-miR-15a. ISIS 356227 (SEQ ID NO: 23) targets aregion flanking and immediately 3′ to the predicted 3′ Drosha cleavagesite in pri-miR-15a. Additionally, oligomeric compound ISIS 327951 (SEQID NO: 30), a uniform 2′-MOE compound targeting mature miR-15a-1, wastested for comparison. Oligomeric compounds ISIS 327901 (SEQ ID NO: 38)targeting mature miR-143; ISIS 129690, (TTAGAATACGTCGCGTTATG; SEQ ID NO:37), a phosphorothioate 5-10-5 MOE gapmer used as a universal scrambledcontrol; and ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 33), auniform 5-10-5 2′-MOE gapmer targeting an unrelated gene, PTEN, wereused as negative controls. Using the transfection methods describedherein, T47D cells were treated with 100 nM of each of these oligomericcompounds. Total RNA was isolated by lysing cells in 1 mL TRIZOL®(Invitrogen, Carlsbad, Calif.) using the manufacturer's recommendedprotocols. Real-time PCR analysis was performed using a primer/probe setspecific for pri-miR-15a [forward primer=ISIS 339317 (SEQ ID NO: 6),reverse primer=ISIS 339318 (SEQ ID NO: 7), and probe=ISIS 339319 (SEQ IDNO: 8)]. Total RNA was quantified using RIBOGREEN® RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.), levels observed for eachtarget were normalized to 5.8S rRNA, and values were expressed relativeto the untreated control (UTC). Effects on expression of pri-miR-15aresulting from treatment of T47D cells with these uniform 2′-MOEoligomeric compounds is expressed as a percentage of RNA levels inuntreated control cells. Results of these experiments are described inTable 3.

TABLE 3 Effects of uniform 2′-MOE oligomeric compounds on pri-miR-15aexpression SEQ ID % ISIS # NO: Target UTC UTC N/A N/A 100 129690 37 N/A121 scrambled control 327901 38 miR-143 132 116847 33 PTEN mRNA 132327951 30 mature miR-15a-1 713 356213 9 >100 bp upstream of mature 171miR-15a 356215 11 flanking 5′ Drosha 1005 cleavage site of pri-miR-15a-1356216 12 pri-miR-15a-1 503 356218 14 loop of pri-miR-15a-1 392 35622117 pri-15a-1 444 356224 20 pri-15a-1 592 356227 23 flanking 3′ Drosha879 cleavage site of pri-15a-1 356229 25 pri-15a-1 818 356231 27pri-15a-1 811 356232 28 pri-15a-1 631

From these data, it was observed that the uniform 2′-MOE oligomericcompounds ISIS 327927, 327951, 356215, 356216, 356218, 356221, 356224,356227, 356229, 356231 and 356232 stimulate an increase in levels ofpri-miR-15a as detected by real-time PCR. Notably, oligomeric compoundsISIS 356215 and 356227, which target the regions immediately flankingthe predicted 5′ and 3′ Drosha cleavage sites in pri-miR-15a,respectively, were observed to stimulate the greatest increases inlevels of pri-miR-15a. It is possible that these oligomeric compoundsbind to pri-miR-15a and/or the respective pre-miRNA and interfere withtheir processing into mature miR-15a, possibly by interfering with theactivity of RNase III-like enzymes such as human Dicer and/or Drosha. Itis understood that such effect of oligomeric compounds may be operatingnot only upon regulation of miR-15a production and processing, but mayalso be found to regulate the production and processing of other miRNAs.

The expression levels of miR-24-2, let-7i, and let-7d were assessed inHeLa or T-24 cells treated with various uniform 2′-MOE oligomericcompounds targeting mature miRNAs (designed using publicly availablemature miRNA sequences). For example, using the transfection methodspreviously described, HeLa cells were treated with 100 nM of theoligomeric compound ISIS 327945 (SEQ ID NO: 39) targeting maturemiR-24-2. Total RNA was isolated, subjected to a reverse transcriptasereaction, and levels of the pri-miR-24-2 were analyzed by quantitativereal-time PCR using a primer/probe set specific for pri-miR-24-2[forward primer=ISIS 359358 (CCCTGGGCTCTGCCT; herein incorporated as SEQID NO: 40), reverse primer=ISIS 359359 (TGTACACAAACCAACTGTGTTTC; hereinincorporated as SEQ ID NO: 41), and probe=ISIS 359360 (CGTGCCTACTGAGC;herein incorporated as SEQ ID NO: 42)]. An approximately 35-foldincrease in levels of pri-miR-24-2 was observed in HeLa cells treatedwith the oligomeric compound ISIS 327945 as detected by real-time PCR.

Using the transfection methods previously described, HeLa cells weretreated with 100 nM of the oligomeric compound ISIS 327890 (SEQ ID NO:43) targeting the mature let-7i. Total RNA was isolated, subjected toreverse transcriptase, and levels of the let-7i pri-miRNA were analyzedby real-time PCR using a primer/probe set specific for the let-7ipri-miRNA [forward primer=ISIS 341684 (TGAGGTAGTAGTTTGTGCTGTTGGT; hereinincorporated as SEQ ID NO: 44), reverse primer=ISIS 341685(AGGCAGTAGCTTGCGCAGTTA; herein incorporated as SEQ ID NO: 45), andprobe=ISIS 341686 (TTGTGACATTGCCCGCTGTGGAG; herein incorporated as SEQID NO: 46)]. An approximately 4-fold increase in levels ofpri-miR-let-7i molecule was observed in HeLa cells treated with theoligomeric compound ISIS 327890 as detected by real-time PCR.

Using the transfection methods previously described T-24 cells weretreated with 100 nM of the oligomeric compound ISIS 327926 (SEQ ID NO:47) targeting mature let-7d. Total RNA was isolated and subjected toreverse transcriptase, and levels of the let-7d pri-miRNA were analyzedby real-time PCR using a primer/probe set specific for let-7d pri-miRNA(forward primer=ISIS 341678 (CCTAGGAAGAGGTAGTAGGTTGCA; hereinincorporated as SEQ ID NO: 48), reverse primer=ISIS 341679(CAGCAGGTCGTATAGTTACCTCCTT; herein incorporated as SEQ ID NO: 49), andprobe=ISIS 341680 (AGTTTTAGGGCAGGGATTTTGCCCA; herein incorporated as SEQID NO: 50)). An approximately 1.7-fold increase in levels of let-7dpri-miRNA was observed in T-24 cells treated with the oligomericcompound ISIS 327926 as detected by real-time PCR.

Thus, treatment with uniform 2′-MOE oligomeric compounds targetingmature miRNAs resulted in the accumulation of pri-miRNA from which themature miRNA is derived.

In one embodiment, the expression of miR-21 (noted to be expressed athigh levels in HeLa cells) was assessed in cells treated with oligomericcompounds. Using the transfection methods previously described, HeLacells were treated with 100 nM of the uniform 2′-MOE oligomeric compoundISIS 327917 (SEQ ID NO: 51) targeting mature miR-21. Total RNA wasisolated by lysing cells in 1 mL TRIZOL® (Invitrogen, Carlsbad, Calif.)using the manufacturer's recommended protocols. By Northern blotanalysis of total RNA from HeLa cells treated with ISIS 327917, levelsof the miR-21 mature miRNA were observed to be reduced to 50% of thoseof untreated control cells. Furthermore, levels of pri-miR-21 were foundto increase in these HeLa cells treated with the oligomeric compoundISIS 327917. Reverse transcriptase and real-time PCR analysis was alsoperformed on RNA isolated from HeLa cells treated with ISIS 327917 usinga primer/probe set specific for pri-miR-21 molecule [forward primer=ISIS339332 (GCTGTACCACCTTGTCGGGT; herein incorporated as SEQ ID NO: 52),reverse primer=ISIS 339333 (TCGACTGGTGTTGCCATGA; herein incorporated asSEQ ID NO: 53), and probe=ISIS 339334 (CTTATCAGACTGATGTTGACTGTTGAAT;herein incorporated as SEQ ID NO: 54)]. Total RNA was quantified usingRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.), levels observed for the target were normalized to 5.8S rRNA, andvalues were expressed relative to an untreated control (UTC). ISIS327917 was observed to stimulate an approximately 2-fold increase inlevels of pri-miR-21 as detected by real-time PCR.

Thus, these data suggest that, in addition to binding the miR-21 maturemiRNA and interfering with the RISC-mediated binding of miR-21 to itsmRNA target, the oligomeric compound, ISIS 327917, may bind topri-miR-21 and/or the respective pre-miRNA and interfere with processingof the mature miR-21, which results in reduction of mature miR-21 levelsin HeLa cells, possibly by interfering with the activity of RNaseIII-like enzymes such as human Dicer or Drosha.

In accordance with the present invention, a nested series of uniform2′-MOE oligomeric compounds were designed and synthesized to target theentire length of pri-miR-21. The oligomeric compounds were designedusing publicly available sequence information (nucleotides 16571584 to16571864 of GENBANK® Accession number NT_(—)010783.14, deposited withGENBANK® on Oct. 7, 2003; SEQ ID NO: 103). Each compound is 20nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotidesand phosphorothioate (P═S) internucleoside linkages throughout thecompound. All cytidine residues are 5-methylcytidines. The compounds areshown in Table 4. The compounds are analyzed for their effect on maturemiRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR;alternatively, they are used in other assays to investigate the role ofmiRNAs or the function of targets downstream of miRNAs.

TABLE 4 Uniform 2′-MOE PS Compounds targeting pri-miR-21 ISIS SEQ IDNumber NO Sequence 358765 55 ACAAGCAGACAGTCAGGCAG 358766 56GGTAGGCAAAACAAGCAGAC 358767 57 GGAGATGTCACGATGGTAGG 358768 58AGGTGGTACAGCCATGGAGA 358769 59 GATAAGCTACCCGACAAGGT 358770 60AGTCTGATAAGCTACCCGAC 358771 61 CAACAGTCAACATCAGTCTG 358772 62GAGATTCAACAGTCAACATC 358773 63 CTGGTGTTGCCATGAGATTC 358774 64CATCGACTGGTGTTGCCATG 358775 65 ACAGCCCATCGACTGGTGTT 358776 66TGTCAGACAGCCCATCGACT 358777 67 CCAAAATGTCAGACAGCCCA 358778 68GATACCAAAATGTCAGACAG 358779 69 GGTCAGATGAAAGATACCAA 358780 70AACATTGGATATGGATGGTC 358781 71 TAATGTTTAAATGAGAACAT 358782 72AACAATGATGCTGGGTAATG 358783 73 GAGTTTCTGATTATAAACAA 358784 74CGACAAGGTGGTACAGCCAT 358785 75 GAAAGATACCAAAATGTCAG

Pri-miR-21 levels were compared in HeLa cells treated with this nestedseries of uniform 2′-MOE oligomeric compounds, targeting and spanningthe entire length of pri-miR-21. ISIS 358768 (SEQ ID NO: 58) targets aregion flanking the predicted 5′ Drosha cleavage site in pri-miR-21.ISIS 358777 (SEQ ID NO: 67) targets a region spanning the 3′ Droshacleavage site in pri-miR-21. ISIS 358779 (SEQ ID NO: 69) targets aregion flanking the predicted 3′ Drosha cleavage site in pri-miR-21.Additionally, oligomeric compounds ISIS 327917 (SEQ ID NO: 51), auniform 2′-MOE compound targeting the mature miR-21 miRNA, and ISIS345382 (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 51), a 5-10-7phosphorothioate 2′-MOE gapmer targeting miR-21, were tested forcomparison. Oligomeric compound ISIS 327863 (ACGCTAGCCTAATAGCGAGG;herein incorporated as SEQ ID NO: 76), a phosphorothioate 5-10-5 2′-MOEgapmer, was used as scrambled control. Using the transfection methodspreviously described, HeLa cells were treated with 100 nM of each ofthese oligomeric compounds. Total RNA was isolated by lysing cells in 1mL TRIZOL® (Invitrogen, Carlsbad, Calif.) using the manufacturer'srecommended protocols, and was subjected to reverse transcriptase.Real-time PCR analysis was performed using the primer/probe set specificfor pri-miR-21 [forward primer=ISIS 339332 (SEQ ID NO.: 52), reverseprimer=ISIS 339333 (SEQ ID NO: 53), and probe=ISIS 339334 (SEQ ID NO:54)]. Total RNA was quantified using RIBOGREEN® RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.), levels observed for eachtarget were normalized to 5.8S rRNA, and values were expressed relativeto the untreated control (UTC). Effects on levels of pri-miR-21resulting from treatment of HeLa cells with these uniform T-MOEoligomeric compounds is expressed as a percentage of RNA levels inuntreated control cells. Results of these experiments are shown in Table5.

TABLE 5 Effects of oligomeric compounds on pri-miR-21 expression SEQ ID% ISIS # NO: Target UTC UTC N/A N/A 100 327863 76 N/A 107 gapmer control327917 51 mature miR-21 249 Uniform 2′-MOE 345382 51 mature miR-21 1195-10-7 2′-MOE gapmer 358765 55 pri-miR-21 133 358766 56 pri-miR-21 142358767 57 pri-miR-21 248 358768 58 flanking 5′ Drosha 987 cleavage siteof pri-miR-21 358769 59 pri-miR-21 265 358770 60 pri-miR-21 250 35877161 pri-miR-21 181 358772 62 pri-miR-21 245 358773 63 pri-miR-21 148358774 64 pri-miR-21 104 358775 65 pri-miR-21 222 358776 66 pri-miR-21367 358777 67 spanning 3′ Drosha 536 cleavage site of pri-miR-21 35877868 pri-miR-21 503 358779 69 flanking 3′ Drosha 646 cleavage site ofpri-miR-21 358780 70 pri-miR-21 269 358781 71 pri-miR-21 122 358782 72pri-miR-21 155 358783 73 pri-miR-21 133 358784 74 pri-miR-21 358 35878575 pri-miR-21 257

From these data, it was observed that the uniform 2′-MOE oligomericcompounds ISIS 327917, 358767, 358768, 358769, 358770, 358772, 358775,358776, 358777, 358778, 358779, 358780, 358784 and 358785 stimulate anincrease in pri-miR-21 levels as detected by real-time PCR. Notably,oligomeric compounds ISIS 358768 and 358779 which target the regionsflanking the predicted 5′ and 3′ Drosha cleavage sites, respectively,and ISIS 358777, which targets a region spanning the 3′ Drosha cleavagesite in pri-miR-21 were observed to stimulate the greatest increases inlevels of pri-miR-21. Furthermore, treatment of HeLa cells withincreasing concentrations (25, 50, 100, and 200 nM) of ISIS 358768,358779, and 327917 was observed to result in a dose-responsive increasein pri-miR-21 levels. Thus, it is believed that these oligomericcompounds bind to pri-miR-21 and/or the respective pre-miRNA andinterfere with their processing into mature miR-21, possibly byinterfering with the activity of RNase III-like enzymes such as humanDicer and/or Drosha. It is understood that such effects of oligomericcompounds may be operating not only upon regulation of miR-21 productionand processing, but may also be found to regulate the production andprocessing of other miRNAs or target nucleic acids.

In one embodiment, the oligomeric compounds ISIS 327917 (SEQ ID NO: 51),the phosphorothioate uniform 2′-MOE targeting mature miR-21; ISIS 358768(SEQ ID NO: 58), the uniform 2′-MOE targeting pri-miR-21 whichstimulated the largest increase in pri-miRNA levels by real time PCR;and ISIS 345382 (SEQ ID NO: 51), the 5-10-7 phosphorothioate 2′-MOEgapmer targeting mature miR-21 were selected for dose response studiesin HeLa cells using the luciferase reporter system described in U.S.Application Publication No. 2005/0261218. ISIS 342683 (SEQ ID NO: 77),representing the scrambled nucleotide sequence of an unrelated PTP1Bantisense oligonucleotide, was used as a negative control. HeLa cellsexpressing the pGL3-miR-21 sensor plasmid (U.S. Application PublicationNo. 2005/0261218) were treated with 1.9, 5.5, 16.7, and 50 nM of theseoligomeric compounds, to assess the ability of oligomeric compounds tointerfere with endogenous miR-21-mediated silencing of the pGL3-miR-21sensor plasmid. The data are presented in Table 6 as percent untreatedcontrol (luciferase plasmid only, not treated with oligomeric compound)luciferase expression, normalized to pRL-CMV levels.

TABLE 6 Effects of oligomeric compounds on miR-21 miRNA- mediatedinhibition of luciferase expression % UTC Dose of oligomeric compoundTreatment 1.9 nM 5.5 nM 16.7 nM 50 nM 342683 127 171 104 108 Negativecontrol 327917 522 1293 2470 4534 358768 103 163 146 118 345382 101 135117 95

From these data, it was observed that, at all doses, treatment of HeLacells with ISIS 327917, the uniform 2′-MOE oligomeric compound targetingthe mature miR-21 miRNA, de-repressed the expression of the luciferasereporter, in a dose-dependent fashion. Thus, ISIS 327917 reversed thesilencing effect of the endogenous miR-21 miRNA, possibly by inhibitingthe binding of miR-21 to its target site encoded by the pGL3-miR-21sensor plasmid.

A similar assay using the miR-21 luciferase reporter system wasperformed, using oligomeric compounds ISIS 327917, ISIS 365241, ISIS358762, ISIS 358758, and ISIS 345382; each of these compounds has thenucleobase sequence TCAACATCAGTCTGATAAGCTA (incorporated herein as SEQID NO: 51). Also tested was ISIS 342683 (CCTTCCCTGAAGGTTCCTCCTT,incorporated herein as SEQ ID NO: 77). ISIS 342683 is not designed tohybridize to miR sequences and was used as a negative control. ISIS327917 and ISIS 342683 are composed of 2′-O-(2-methoxyethyl), also knownas 2′-MOE, nucleotides throughout, and the internucleoside (backbone)linkages are phosphorothioate (P═S) throughout (MOE-PS). ISIS 365241 iscomposed of 2′-MOE nucleotides throughout, and the backbone linkages arephosphodiester (P═O) throughout (MOE-PO). ISIS 358762 is composed of2′-β-methyl residues throughout, and the backbone linkages arephosphorothioate (P═S) throughout (MOE-PS). ISIS 358758 is composed of2′-O-methyl residues throughout, and the backbone linkages arephosphodiester (P═O) throughout (MOE-PO). ISIS 345382 is a chimericoligonucleotide comprised of 10 2′-deoxynucleotides, flanked on the5′-end by 5 2′-MOE nucleotides and on the 3′-end by 7 2′-MOEnucleotides. All cytidine residues in are 5-methylcytidines.

The luciferase assay is described in U.S. application Ser. No.10/909,125. HeLa cells were transiently transfected using LIPOFECTAMINE®2000 transfection reagent (Invitrogen, Carlsbad, Calif.) with pRL-CMVRenilla luciferase plasmid (Promega, Madison, Wis.) and pGL3-miR 21sensor for 24 hours. The pGL3-miR 21 sensor plasmid was prepared usingstandard cloning techniques, such as those described in U.S. ApplicationPublication No. 2005/0261218. Cells were replated and antisenseoligonucleotides targeting mature miR-21, or control antisenseoligonucleotides, were transfected at doses of 1.56, 3.1, 6.25, 12.5,25, or 50 nM for a period of six hours, after which Renilla luciferaseactivity was measured. In a similar experiment, the antisenseoligonucleotides, at doses of 1.56, 3.1, 6.25, 12.5, 25, or 50 nM, weretransfected simultaneously with the plasmids for a period of 24 hours,after which Renilla luciferase activity was measured. Theoligonucleotides were evaluated for their ability to interfere withmiR-21 inhibition of pGL3-miR-21 sensor luciferase expression. Eachtreated sample was normalized to untreated control samples.

This assay revealed that while compounds with either the 2′-MOEmodification or the 2′-O-methyl modification can interfere with miR-21activity in a dose-dependent fashion, oligomeric compounds having 2′-MOEmodifications exhibited an increased ability to inhibit miR-21 activityafter 24 hours of treatment, as compared to after 6 hours of treatment.

Example 2 Effect of Oligomeric Compounds Targeted to pri-miR-15a onLevels of Related or Unrelated miRNAs

Members of miRNA families are characterized by the presence of anidentical seed sequence. In a further embodiment, oligomeric compoundstargeted to positions of pri-miR-15a were tested for their effects onrelated and unrelated pri-miRs. The nucleobase sequences of members ofthe miR-15 family are shown in Table 7. Seed sequences within eachmature miRNA sequence are underlined.

TABLE 7 miR-15 family members SEQ Mismatches ID relative to Sequence NO:miRNA miR-15a -UAGCAGCACAUAAUGGUUUGUG--- 98 miR-15a 0-UAGCAGCACAUCAUGGUUUACA--- 99 miR-15b 4 -UAGCAGCACGUAAAUAUUGGCG--- 100miR-16 6 -UAGCAGCACAGAAAUAUUGGC---- 101 miR-195 6

Oligomeric compounds were designed using publicly available sequence(the complement of nucleotides 31603159 to 31603468 of GENBANK®Accession number NT_(—)024524.13, deposited with GENBANK® on Oct. 7,2003). HeLa cells were treated with the compounds in Table 8 for aperiod of 16 to 20 hours. RNA was isolated from the treated cells usingTRIZOL® (Invitrogen, Carlsbad, Calif.) and TURBO™ DNase (Ambion, Austin,Tex.), and was subjected to a reverse transcription reaction, which wasfollowed by real-time PCR with primer probe sets designed to detectpri-miR-15a, pri-miR-16-1, pri-miR-15b and pri-miR-21.

Primers and probe to detect miR-15a are:

(SEQ ID NO: 6) Forward primer, GTGTGTTTAAAAAAAATAAAACCTTGGA(SEQ ID NO: 7) Reverse primer, TGGCCTGCACCTTTTCAAA (SEQ ID NO: 8)Probe, AAAGTAGCAGCACATAATGGTTTGTGG

Primers and probe to detect miR-16-1 are:

(SEQ ID NO: 78) Forward primer, CAATATTTACGTGCTGCTAAGGCA (SEQ ID NO: 79)Reverse primer, CAACCTTACTTCAGCAGCACAGTT (SEQ ID NO: 80)Probe, CTGGAGATAATTTTAGAATCTTAACG

Primers and probe to detect miR-15b are:

(SEQ ID NO: 81) Forward primer, CAGTACTTTAAGGCCTCAAAAATGTAGG(SEQ ID NO: 35) Reverse primer, CAAATAATGATTCGCATCTTGACTGT(SEQ ID NO: 82) Probe, GCATGTAAACCATGATGTGCTGCT

Primers and probe to detect miR-21 are:

(SEQ ID NO: 83) Forward primer, ACCCGACAAGGTGGTACAGC (SEQ ID NO: 53)Reverse primer, TCGACTGGTGTTGCCATGA (SEQ ID NO: 84)Probe, ATTCAACAGTCAACATCAGTCTGATAAG

As indicated in the “Chemistry” column in Tables 8, “5-10-5” compoundsare chimeric oligomeric compounds, composed of a gap segment 10nucleotides in length which is comprised of 2′-deoxynucleotides. The“wing” segments on either side (5′ and 3′) of the oligomeric compoundare comprised of five 2′-O-(2-methoxyethyl), also known as 2′-MOE,nucleotides. “5-10-7” compounds are chimeric oligomeric compounds,composed of a gap segment 10 nucleotides in length which is comprised of2′-deoxynucleotides. The 5′ wing segments is comprised of five 2′-MOEnucleotides and the 3′ wing segment is comprised of seven 2′-MOEnucleotides. “Full 2′-MOE” oligomeric compounds are comprised of 2′-MOEnucleotides throughout. In all compounds in Tables 1 and 2,internucleoside (backbone) linkages are phosphorothioate (P═S)throughout, and all cytidine residues are 5-methylcytidines. In Table 8,“Drosha” indicates a Drosha recognition region; “MM” indicates aoligonucleotide having mismatches to a target sequence; “NC” indicatesnegative control; “Extended” is used to describe oligomeric compoundsthat are outside Drosha recognition regions; “Target” indicates theparticular mature miRNA targeted by the oligomeric compound; and“Position on pri-miR-15a” indicates the site to which the oligomericcompounds are targeted on the pri-miR-15 a sequence (the complement ofnucleotides 31603159 to 31603468 of GENBANK® Accession numberNT_(—)024524.13, deposited with GENBANK® on Oct. 7, 2003).

Shown in Table 9 are levels observed for each target were normalized to5.8S rRNA, and values were expressed relative to the untreated control(UTC). Each treatment was performed in duplicate.

TABLE 8 Oligomeric compounds targeted to pri-miR-15a SEQ Position IDon pri- Isis # SEQUENCE Motif NO: Target Target Region miR-15a 345391TGTAAACCATGATGTGCTGCTA 5-10-7 29 miR- None None 15b 347964TATAACATTGATGTAATATG 5-10-5 9 miR- Gapmer of 12 15a 356213 NC, Extended347966 TGCTACTTTACTCCAAGGTT 5-10-5 11 miR- Gapmer of 129 15a 356215Drosha 345411 CACAAACCATTATGTGCTGCTA 5-10-7 30 miR- Mature miR 144 15a347980 AGATCAGATCCTTGTATTTT 5-10-5 25 miR- Gapmer of 203 15a 356229Drosha 327901 TGAGCTACAGTGCTTCATCTCA Full 2′- 38 miR- None None MOE 143356213 TATAACATTGATGTAATATG Full 2′- 9 miR- NC, Extended 12 MOE 15a360644 GAATGCATGTAAAAAAATCT Full 2′- 85 miR- Extended 44 MOE 15a 360643TCTTTCAGGAAAAAAAGAAT Full 2′- 86 miR- Extended 60 MOE 15a 360642AATATAAAAAATATTTTCTT Full 2′- 87 miR- Extended 76 MOE 15a 360641ACATTCGCGCCTAAAGAATA Full 2′- 88 miR- Extended 92 MOE 15a 360640TATTTTTTTTAAACACACAT Full 2′- 89 miR- Extended 108 MOE 15a 360639CTTTACTCCAAGGTTTTATT Full 2′- 90 miR- Extended 124 MOE 15a 356215TGCTACTTTACTCCAAGGTT Full 2′- 11 miR- Drosha 129 MOE 15a 327951CACAAACCATTATGTGCTGCTA Full 2′- 30 miR- Mature miR 144 MOE 15a 356225TATTTTTGAGGCAGCACAAT Full 2′- 21 miR- Drosha 189 MOE 15a 356228AGATCCTTGTATTTTTGAGG Full 2′- 24 miR- Drosha 198 MOE 15a 356229AGATCAGATCCTTGTATTTT Full 2′- 25 miR- Drosha 203 MOE 15a 356230AGAAGATCAGATCCTTGTAT Full 2′- 26 miR- Drosha 206 MOE 15a 356231TTCAGAAGATCAGATCCTTG Full 2′- 27 miR- Drosha 209 MOE 15a 356232AAATATATTTTCTTCAGAAG Full 2′- 28 miR- Drosha 221 MOE 15a 360647TAAGAGCTATGAATAAAAAG Full 2′- 91 miR- Extended 241 MOE 15a 360648GCTGACATTGCTATCATAAG Full 2′- 92 miR- Extended 257 MOE 15a 371778CTAAGGCACTGCTGACATTG Full 2′- 93 miR-16-1 Drosha 267 MOE 360649GTGCTGCTAAGGCACTGCTG Full 2′- 94 miR- Extended 273 MOE 15a 360650TAACGCCAATATTTACGTGC Full 2′- 95 miR- Extended 289 MOE 15a 371783GTAGAGTATGGTCAACCTTACT Full 2′- 96 miR16-1 Drosha 348 MOE 340927TGAGCTACAGTGCTTCATCTCA 5-10-7 38 miR- None None 143 327927TGTAAACCATGATGTGCTGCTA Full 2′- 29 miR- None None MOE 15b 342682CCTTCCCTGAAGGTTCCTCCT Full 2′- 97 MM None None MOE

TABLE 9 Pri-miR-15a, -15b, -16-1, and -21 levels following treatment ofHeLa cells with oligomeric compounds targeted to pri-miR-15a SEQPosition pri- pri- pri- pri- ID on pri- mir- miR- miR- miR- ISIS # NO:Target Target Region miR-15a 15a 21 15b 16-1 345391 29 miR-15b None None0.61 0.9 0.4 1.6 347964 9 miR-15a Gapmer of 356213 12 0.27 1.2 0.5 0.5NC, Extended 347966 11 miR-15a Gapmer of 356215 129 0.14 1.7 0.4 0.4Drosha 345411 30 miR-15a Mature miR 144 0.38 0.9 0.8 2.8 347980 miR-15aGapmer of 356229 203 0.93 0.9 0.5 0.7 Drosha 327901 38 miR-143 None None0.67 1.0 1.0 1.2 356213 9 miR-15-a NC, Extended 12 0.83 1.7 0.5 0.4360644 85 miR-15-a Extended 44 0.60 1.5 0.9 0.2 360643 86 miR-15-aExtended 60 0.65 1.4 0.8 0.9 360642 87 miR-15-a Extended 76 0.51 1.4 0.50.8 360641 88 miR-15-a Extended 92 1.25 1.9 2.2 1.2 360640 89 miR-15-aExtended 108 0.99 1.4 0.9 0.6 360639 90 miR-15-a Extended 124 2.68 1.00.8 0.8 356215 11 miR-15a Drosha 129 3.45 1.3 1.1 0.9 327951 30 miR-15aMature miR 144 3.70 1.3 3.3 8.3 356225 21 miR-15a Drosha 189 0.61 1.60.6 0.7 356228 24 miR-15a Drosha 198 2.44 1.2 0.6 0.7 356229 25 miR-15aDrosha 203 2.08 2.1 0.6 0.4 356230 26 miR-15a Drosha 206 2.40 0.9 0.80.9 356231 27 miR-15a Drosha 209 2.40 1.3 0.9 1.0 356232 28 miR-15aDrosha 221 2.05 1.7 0.5 0.8 360647 91 miR-15a Extended 241 0.62 1.7 0.81.1 360648 92 miR-15a Extended 257 1.84 0.9 0.3 15.1 371778 93 miR-16-1Drosha 267 2.49 0.8 0.6 15.0 360649 94 miR-15a Extended 273 3.00 1.6 1.023.9 360650 95 miR-15a Extended 289 3.45 0.9 1.1 15.2 371783 96 miR-16-1Drosha 348 0.81 1.2 0.3 1.3 340927 38 miR-143 None None 0.62 1.0 0.7 0.7327927 29 miR-15b None None 2.12 0.9 1.9 5.9 342682 97 None None None0.85 1.5 1.1 1.0

Oligomeric compounds targeting mature miR-15a and mature miR-16 resultedin the highest increases in pri-mi-miR-15 and pri-miR-16 levels RNAlevels. The regions up to 16 nucleotides in the 5′ direction relative tothe 5′ end of either the mature miR-15a sequence or the mature miR-16sequence, and up to 40 nucleotides in the 3′ direction relative to the3′ end of mature miR-15a, also resulted in increased pri-miRNA levels.An increase in pri-miR-15 a levels was seen with oligomeric compoundstargeting the hairpin region of pri-miRNA, however, oligomeric compoundstargeting the stem of the hairpin were more effective at increasingpri-miRNA levels as compared to oligomeric compounds targeting the loopregion. Oligomeric compounds targeting mature miR-15a caused an increasein the levels of related pri-miR-15a, pri-miR-15b and pri-miR-16.Oligomeric compounds targeting Drosha recognition regions did not causethe levels of a non-related pri-miRNA, pri-miR-21, to increase.

Oligomeric compounds targeting Drosha recognition regions affectedpri-miR-15a and pri-miR-16-1 levels in different manners. For example,oligomeric compounds targeting the Drosha recognition region of miR-15adid not affect pri-miR-16-1 levels. Conversely, oligomeric compoundstargeting Drosha recognition regions on pri-miR-16-1 increasedpri-miR-16-1 and pri-miR-15a levels. It is known that miR-16 ispreferentially processed relative to miR-15a, thus these data suggestthat targeting the Drosha recognition region of a preferentiallyprocessed mature miRNA is a means by which a single oligomeric compoundcan be used to modulate the levels of multiple, related miRNAs.

Example 3 Effects of Oligomeric Compounds Targeting pri-miRNAs andMature miRs in vivo

In a further embodiment, oligomeric compounds were tested in vivo toevaluate the effects of compounds targeting mature miRNAs, as well asthe effects of oligomeric compounds which target Drosha cleavage sites.The miRNAs and pri-miRNAs selected were those corresponding to miR-15aand miR-21. The oligomeric compounds targeting the miR-15a mature miRNA.The oligomeric compounds targeting the mature miR-21 and Drosha cleavagesite are ISIS 327917 and ISIS 358779 (GGTCAGATGAAAGATACCAA, SEQ ID NO:69), respectively. These 4 compounds are uniformly comprised of 2′-MOEnucleotides and have phosphorothioate (P═S) internucleoside linkagesthroughout. All cytidine residues are 5-methylcytidines

Male Balb/c mice, obtained from The Jackson Laboratories (Ben Harbor,Me.), were fed a standard rodent diet and were injected with 25 mg/kg ofISIS 327917, ISIS 358779, ISIS 327951 or ISIS 356415. Each treatmentgroup contained a total of 5 animals. A control group received salineinjections only. Injections were administered intraperitoneally, twiceweekly for a period of 4 weeks. Animals were sacrificed after the finaldose, and RNA was prepared from liver tissue. miR levels were measuredby Northern blotting and normalized to U6 RNA, as described herein. Thedata are shown in Table 9 as the average of each treatment group.

TABLE 9 In vivo testing of oligomeric compounds targeting mature miRsand Drosha cleavage sites miR levels normalized to U6 RNA miR-21 miR-15aSaline 0.46 17.7 ISIS 327917 (mature miR-21) 0.33 19.5 ISIS 358779(miR-21, Drosha cleavage site) 0.38 15.1 ISIS 327951 (mature miR-15a)0.84 9.4 ISIS 356215 (miR-15a, Drosha cleavage site) 0.66 8.7

These data demonstrate that with regard to miR-21 and miR-15a,oligomeric compounds targeting Drosha cleavage sites and oligomericcompounds targeting mature miRNAs reduce miRNA expression to similarlevels in vivo.

What is claimed:
 1. A method of increasing the amount of pri-miR-21 in acell comprising: contacting the cell with an oligomeric compoundcomprising an oligonucleotide consisting of 15 to 30 linked nucleosidesand having a nucleobase sequence selected from SEQ ID NOs: 57 to 60, 65to 70, and 74, whereby the amount of pri-miR-21 in the cell isincreased.
 2. The method of claim 1 wherein the oligonucleotide consistsof 17 to 25 linked nucleosides.
 3. The method of claim 1 wherein theoligomeric compound consists of an oligonucleotide.
 4. The method ofclaim 1 wherein at least one nucleoside of the oligonucleotide comprisesa sugar modification.
 5. The method of claim 4 wherein each nucleosideof the oligonucleotide comprises a sugar modification.
 6. The method ofclaim 5 wherein the sugar modification is 2′-O-methoxyethyl.
 7. Themethod of claim 1 wherein at least one internucleoside linkage is amodified internucleoside linkage.
 8. The method of claim 1 wherein eachinternucleoside linkage is a modified internucleoside linkage.
 9. Themethod of claim 8 wherein the modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 10. The method of claim 1wherein the oligonucleotide comprises at least one cytosine, wherein thecytosine is a 5-methylcytosine.
 11. The method of claim 10 wherein eachcytosine is a 5-methylcytosine.